US20180327770A1 - Guayule with increased rubber production and yield

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Abstract

Description

Claims

US20180327770A1

Publication number: US20180327770A1
Application number: US15/975,912
Authority: US
Inventors: Colleen M. McMahan; Dante Placido; David Dierig; Von Mark Cruz
Original assignee: Bridgestone Corp; US Department of Agriculture USDA
Current assignee: Bridgestone Corp; US Department of Agriculture USDA
Priority date: 2017-05-11
Filing date: 2018-05-10
Publication date: 2018-11-15
Also published as: EP3622077A1; EP3622067A4; EP3622067A1; MX2019013391A; US20210238619A1; US10982219B2; MX2019013413A; WO2018209176A1; US20180327765A1; WO2018209184A1; EP3622077A4

A reduction in the amount of functional PaAos in guayule results in the production of increased amounts rubber compared to the amount of rubber produced by wild-type guayule having a non-reduced amount of functional PaAos. Further, the guayule with reduced amount of functional PaAos are larger than wild-type guayule and thus have larger rubber yield per acre than wild-type guayule. Reduction of the amount of functional PaAos in guayule can be caused by genetic alterations in PaAos. Guayule having PaAos with a specific amino acid sequence produces more rubber than guayule with PaAos having a different amino acid sequence. Thus, one can use the sequence differences as a biomarker for selecting high rubber producing guayule plants.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Patent Application 62/504,762 filed on May 11, 2017, contents of which are expressly incorporated by reference herein.

BACKGROUND OF THE INVENTION

Sequence Listing

The Sequence Listing submitted via EFS-Web as ASCII compliant text file format (.txt) filed on May 10, 2018, named “SequenceListing_ST25”, (created on May 9, 2018, 24 KB), is incorporated herein by reference. This Sequence Listing serves as paper copy of the Sequence Listing required by 37 C.F.R. § 1.821(c) and the Sequence Listing in computer-readable form (CRF) required by 37 C.F.R. § 1.821(e). A statement under 37 C.F.R. § 1.821(f) is not necessary.

Field of the Invention

This invention relates to altered guayule plants that grow larger and produce more rubber than non-altered guayule plants, when grown under the same conditions. The altered guayule contain the cDNA sequence of Parthenium argentatum Allene oxide synthase (PaAos) in the reverse complement orientation under control of a heterologous promoter which reduces the production of PaAos via RNAi. Other types of genetic alternations can be made in guayule to reduce the functionality of PaAos. Kits for identifying such altered guayule, methods for identify the altered guayule, and methods of increasing rubber yield in guayule via reducing PaAos translation are also included.

Description of Related Art

Natural rubber is synthesized by more than 2,500 plant species (Cornish, et al., J. Nat. Rubber Research 8:275-285 (1993); Cornish, K., Phytochemistry 57:1123-1134 (2001)). Rubber is produced by these plants as a secondary metabolite with no clear indication of its function in plant cells. Possible reasons on why these species synthesize rubber are to defend themselves against pathogens and insect attacks, repair tissue damages caused by mechanical wounding and protect cell damage induced by environmental stresses (Demel, et al., Biochim. Biophys. Acta. 1375:36-42 (1998); Tangpakdee and Tanaka, J. Rubber Res. 1:14 (1998); Vereyken, et al., Biochim. Biophys. Acta, 1510:307-320 (2001); Kim, et al., Plant Cell Physiol., 412-414 (2003) and references therein; Konno, K., Phytochemistry, 1510-1530 (2011); and Sarkar, J., Rubber Science, 228-237 (2013)). According to a 2014 market report, the rubber that these plants produce accounted for $16.5 billion in trade worldwide (rubberworld.com/RWmarket_report.asp). Even more so, the end products made from natural rubber, including tires for the transportation industry, sports equipment, medical devices, and more, are indispensable in our everyday life. The Hevea tree is the main source of natural rubber but concerns exist as it is limited geographically to tropical climates, mainly in Southeast Asia, is susceptible to diseases, and produces rubber that causes allergic reactions. Clearly, an alternative source for the production of natural rubber is very important to reduce economic risk and safeguard human health.
One plant known to be a promising source of natural rubber is guayule (Parthenium argentatum, Gray), a desert shrub native to the southwestern United States and northern Mexico (Mooibroek and Cornish, Appl. Microbio. and Biochem. 53:355-365 (2000); van Beilen and Poirier, Critical Reviews Biotech. 27:217-231 (2007)). The majority of rubber synthesis in guayule occurs during the cold season. Guayule synthesizes rubber within subcellular organelles called rubber particles (Archer and Audley, Bot. J. Linnean Soc. 94:181-196 (1987)) stored in the parenchyma cells of stembark tissues (Gilliland, M. v., Protoplasma, 169-177 (1984)); Macrae, S. G., Plant Physiol., 1027-1032 (1986)). Natural rubber synthesis is initiated by the action of allylic pyrophosphates initiators (Cornish and Siler, J. Plant Physiol., 301-305 (1995)), usually farnesyl pyrophosphate (FPP). Then, the monomer isopentenyl-pyrophosphate (IPP), produced by the mevalonic acid pathway (MEV) in the cytosol and the methylerythritol phosphate (MEP) pathway in the plastid (Mooibroek and Cornish (2000); van Beilen and Poirier, TRENDS in Biotech., 522-529 (2007)) elongates the rubber chain. Rubber synthesis is mediated by rubber transferases requiring magnesium ions as cofactor (Da Costa, et al., Phytochemistry 67(15): 1621-1628 (2006)).
The proposed model for the structure of rubber particles consists mostly of hydrophobic cis-polyisoprene units (natural rubber) encapsulated inside a protein and phospholipid surface monolayer (Nawamawat, et al., Colloids and Surfaces A: Physicochemical and Engineering Aspects, 390:157-166 (2011); Sansatsadeekul, et al., J. Biosci. and Bioeng, 111:628-634 (2011)). The phospholipids serve to stabilize and solubilize the otherwise insoluble (rubber) product. Guayule rubber particles include several proteins (Whalen, et al., Development of crops to produce industrially useful natural rubber. Chapter 23 in Isoprenoid Synthesis in Plants and Microorganisms: New Concepts and Experimental Approaches, Bach and Rohmer (eds.), DOI 10.1007/978-1-4614-4063-5_23, Springer Science+Business Media NY, 329-345 (2013)) of which Aos has been found to be the most abundant (Backhaus, et al., Phytochemistry 30:2493-2497 (1991)). Aos is well-known as an enzyme in the jasmonic acid biosynthetic pathway (Harms, et al., Plant Cell, 1645-1654 (1995); Wang, et al., Plant Mol. Biol., 783-793 (1999); Schaller, F., J. Exper. Botany, 11-23 (2001)). The role of Aos in rubber biosynthesis, and the reason for the abundance of Aos protein on guayule rubber particle surfaces, is not known (Whalen, et al. (2013)).
The need exists for a method to increase rubber production in altered guayule compared to rubber production amounts in non-altered guayule in order to improve the commercial attractiveness of using guayule rubber as a replacement of synthetic rubber and Hevea rubber. Further, a need exists for increasing the rubber yield per acre obtained from altered guayule compared to the rubber yield per acre obtained from non-altered guayule. This greater rubber yield results from the altered guayule being larger in size than non-altered guayule of similar age. A need also exists for altered guayule that produce more rubber than the amount of rubber produced by non-altered guayule. A need also exists for altered guayule that have a larger size than similarly aged non-altered guayule because the altered guayule that are larger than the non-altered guayule will possess more tissue for storage of rubber and thus generate greater rubber yield per acre than the rubber yield per acre of wild-type guayule. And a need exists for biomarkers which distinguish between low rubber producing and high rubber producing guayules.

BRIEF SUMMARY OF THE INVENTION

It is an object of this invention to have altered guayule, parts of altered guayule, and progeny of the altered guayule, the altered guayule containing a mutation that causes the altered guayule to produce more rubber than the amount of rubber produced by a non-altered guayule. It is a further object of this invention that the mutation in the altered guayule can be one or more of (i) an alteration in a Parthenium argentatum Allene oxide synthase (PaAos) codon encoding an amino acid to a stop codon, (ii) an alteration of PaAos' translation initiation codon to another codon, (iii) an alteration in PaAos ribosome binding site's sequence, (iv) an alteration in one or more PaAos splice site codons, (v) a deletion of part or all of PaAos' sequence, (vi) an insertion of DNA into PaAos, and (vii) an alteration in one or more PaAos DNA codon sequences to encode a non-conservative amino acid. It is an object of this invention that each of these mutations reduces the altered PaAos' functionality compared to the amount of PaAos functionality in a non-altered guayule and that the reduced PaAos functionality causes the altered guayule to produce an increased amount of rubber compared to the amount of rubber produced by the non-altered guayule. It is another object of this invention that the alteration of one or more PaAos DNA codon sequences to encode a non-conservative amino acid occurs at amino acids located at 318, 332, 336, 339, 359, 408, 411, and 459 with PaAos' sequence (see SEQ ID NO: 10, 13, and 15). It is a further object of this invention that the amino acids being changed to non-conservative amino acids are D318, S332, E336, R339, S359, I408, S411, and/or L459. Another object of this invention is that the non-conservative amino acid substitutions are not N318, V408 and/or W459. It is another object of this invention to have an altered cell, germplasm, and an altered seed of the altered guayule, each containing the mutation.
It is an object of this invention to have a method of producing an altered guayule that contains a mutated PaAos and produces more rubber compared to the amount of rubber produced by a non-altered guayule. It is another object of the invention that the method involves exposing a non-altered guayule cell or seed to a mutagen to produce a mutated guayule cell or seed with the mutated PaAos, selecting one or more of the mutated guayule cells or seeds containing the mutated PaAos which encodes an altered PaAos with reduced functionality compared to a non-altered PaAos's functionality, and growing the selected mutated guayule cell or seed containing the mutated PaAos to produce an altered guayule that produces the altered PaAos with reduced functionality and an increased amount of rubber compared to the amount of rubber produced by the non-altered guayule. It is another object of this invention that the mutated PaAos contains at least one of (i) an alteration of a PaAos codon encoding an amino acid to a stop codon, (ii) an alteration of PaAos' translation initiation codon to another codon, (iii) an alteration of PaAos ribosome binding site's sequence, (iv) an alteration of one or more PaAos splice site codons, (v) a deletion of part or all of PaAos' sequence, (vi) an insertion of DNA into PaAos, and (vii) an alteration of one or more PaAos codon sequences to encode a non-conservative amino acid. It is another object of this invention that the alteration of one or more PaAos codon sequences to encode a non-conservative amino acid occurs at amino acids located at 318, 332, 336, 339, 359, 408, 411, and 459 with PaAos' sequence (see SEQ ID NO: 10, 13, and 15). Another object of this invention is that the non-conservative amino acid substitutions are not N318, V408 and/or W459. It is another object of this invention to have an altered cell, germplasm, and an altered seed of the altered guayule produced by this method and each containing the mutation.
It is an object of this invention to have a method for producing a population of high rubber producing guayule plants or seeds which contain PaAos with low functionality. It is an object of that method involves genotyping a first population of guayule plants or seeds that contain PaAos with low functionality, selecting from the first population one or more guayule plants or seeds containing PaAos with low functionality based the genotyping, and producing from the selected one or more guayule plants or seeds containing PaAos with low functionality a second population of guayule plants or seeds containing PaAos with low functionality. It is another object of this invention PaAos with low functionality contains at least one amino acid selected from group of N318, V408, W459, conservative amino acids substitutions thereof, and non-conservative amino acid substitutions at S332, E336, R339, S359, and S411.
It is an object of this invention to have a method of identifying a high rubber producing guayule by detecting the presence of PaAos having amino acids N318, V408 and/or W459, or conservative amino acid substitution at one or more of these positions and/or having non-conservative amino acid substitutions at S332, E336, R339, S359, and S411 in a test guayule. It is further object of this invention that the method involves contacting the PaAos from the test guayule with monoclonal antibodies that binds to PaAos having the above mentioned amino acids, and determining if the monoclonal antibodies bind to the PaAos from the test guayule, where binding indicates the presence of the high rubber producing PaAos and no binding indicates the present of the low rubber producing PaAos. It is another object of this invention that the invention involves obtaining nucleic acids from the test guayule, performing a PCR assay with the obtained nucleic acids, a label, and primer sets SEQ ID NOs: 22 and 23, and SEQ ID NOs: 24 and 25, or similar sequences that encode conservative amino acid substitutions, and determining if an amplicon is generated; such that when an amplicon is produced, then the test guayule contains a PaAos nucleic acid sequence that encodes PaAos having amino acids N318, V408 and/or W459, or one or more of the conservative amino acid substitution at these positions, and/or non-conservative amino acid substitutions at S332, E336, R339, S359, and S411, and the test guayule is a high rubber producing guayule.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the design of the plasmids used in Example 1 for the overexpression of PaAos (pND6-Aos), silencing of PaAos (pND6-AosiL), and for the control plasmid (pND6). Each expression vector features the NPTII gene to confer kanamycin resistance for selection, and the control plasmid contains the GUS (β-glucuronidase) reporter gene instead of PaAos or a portion of PaAos in reverse complementary orientation. Thus, one can use a histochemical GUS staining assay (the chromogenic substrate X-Gluc (C₁₄H₁₃BrCINO₇)) as a visual indicator of transformed tissues with the negative control plasmid.

FIG. 2 shows the primers used in the qRT-PCR, PCR reaction conditions, and the expected amplicon product size to determine RNA expression in the genetically altered guayule. Amplicon (mRNA product) PaAos_OEis derived from plants transformed with pND6-Aos (Aos overexpression). Amplicon (mRNA product) PaAos_RNAiis derived from plants transformed with pND6-AosiL (Aos silencing). Amplicon (mRNA product) 18S is from 18S ribosome RNA. Amplicon (mRNA product) PaAos is for wild-type guayule plants with intact PaAos gene.

FIG. 3 provides size and weight measurements of the four types of guayule plants grown under different conditions in growth chambers. Plants are initially transferred to soil from tissue culture media and grown under greenhouse conditions for one month. Following, plants are moved to controlled-temperature growth chamber conditions under 27° C. (16 h)/25° C. (8 h) and at 27° C. (16 h)/10° C. (8 h). The four type of guayule plants are wild-type (G7-11.1 and G7-11.2), guayule transformed with the empty expression vector pND6 (pND6-10, pND6-12, pND6-35), guayule transformed with pND6-AosiL for silencing PaAos via RNAi (pND6-AosiL_7-2, pND6-AosiL_8-1, pND6-AosiL_9-16, pND6-AosiL_12-1,), and guayule transformed with pND6-Aos to overexpress PaAos (pND6-Aos_4-1, pND6-Aos_4-2, pND6-Aos_5-1, pND6-Aos_7-1,) at 2 months old. G7-11 is a breeder's nomenclature for what later became the USDA publicly released guayule Germplasm line AZ-2 (Reg. No. GP-9; PI 599676). The biomass of the shoot (leaves plus stems) and root are weighed in 2 months old guayule plants grown in growth chambers under 27° C. (16 h)/25° C. (8 h) and at 27° C. (16 h)/10° C. (8 h). The asterisks, (*), (**) and (***), indicate significant difference in comparison to (non-altered) G7-11 at p>0.05, 0.005 and 0.0005, respectively.

FIG. 4 shows SPAD values indicating leaf chlorophyll concentration (“SPAD units”) for wild-type guayule (G7-11), guayule transformed with the empty expression vector (pND6); guayule transformed with pND6-Aos (Oe), and guayule transformed with pND6-AosiL (RNAi) grown at 27° C. (16 h)/25° C. (8 h) or 27° C. (16 h)/10° C. (8 h).

FIG. 5 shows the number of branches and stem diameter of 2 months old genetically altered guayule (pND6-AosiL and pND6-Aos lines), non-altered guayule (G7-11) and empty vector control guayule (pND6) plants grown in growth chambers at 27° C. (16 h)/25° C. (8 h) or at 27° C. (16 h)/10° C. (8 h). pND6-AosiL genotypes have larger number of stems than the non-altered and empty vector controls. Additionally, the mature stembark tissues in pND6-AosiL have significantly thicker diameter (ranging from 35% to 54%) under both 27° C. (16 h)/25° C. (8 h) and 27° C. (16 h)/10° C. (8 h).

FIG. 6 shows results from gel permeation chromatography for elution of cyclohexane extractables from transformed and non-altered guayule plant lines. The natural rubber molecular weight is calculated using Astra software for three pND6-AosiL transformed guayule plants, three pND6-Aos transformed guayule plants, two pND6 transformed guayule plants, and non-altered guayule G7-11. The error bars represent 3 different plants with 3 technical replicates.

FIG. 7 shows the relative expression of PaAos in guayule line G7-11, guayule line W6 549 (“W6549”), and guayule line PI 478652 (“478652”).

FIG. 8A, FIG. 8B, and FIG. 8C show single nucleotide polymorphisms (SNPs) in PaAos coding sequence for guayule cultivars W6 549 (“W6549”; SEQ ID NO: 12), G7-11 (SEQ ID NO: 9), and PI 478652 (“478652”; SEQ ID NO: 14). The SNPs are contained in boxes.

FIG. 9 shows an alignment of PaAos amino acid sequences obtained from guayule cultivars W6 549 (“W6549”; SEQ ID NO: 13), PI 478652 (“478652”; SEQ ID NO: 15) and G7-11 (SEQ ID NO: 10). The boxes highlight the different amino acids in the cultivars.

DETAILED DESCRIPTION OF THE INVENTION

This invention involves the discovery that a reduction in the amount of functional PaAos in guayule results in an increase in the amount of rubber produced. Further, different cultivars of guayule, having different DNA and amino acid sequences for PaAos and PaAos, respectively, produce different amounts of rubber. As such, one can distinguish between guayule cultivars that are “high” rubber producers and “low” rubber producers based on the differences in the DNA and/or amino acid sequence of PaAos and/or PaAos. Because single nucleotide polymorphisms (SNPs) exist in these different cultivars, one can use primers and PCR techniques to determine if any particular guayule is a “high” rubber producing guayule or a “low” rubber producing guayule. Alternatively, one can use antibodies that bind to the different PaAos proteins to determine if any particular guayule is a “high” rubber producer or a “low” rubber producer. As discussed below, guayule cultivar W6 549 has the lowest average rubber content (%) and guayule cultivar PI 478652 has the highest average rubber content, among the tested cultivars. PaAos from both cultivars have slight differences in DNA sequences which, along with the differences in the amino acid sequences, can be used to determine if any particular cultivar is a high or low rubber producer. Any guayule that produces PaAos with conservative amino acid changes to SEQ ID NO: 15 is a high rubber producing guayule; any guayule that produces PaAos with conservative amino acid changes to SEQ ID NO: 13 is a low rubber producing guayule. Thus, one can screen plants for PaAos with conservative sequences to SEQ ID NO: 15 via DNA or protein assays. Alternatively, one screening for guayule with PaAos with the similar (or lower) level of functionality as guayule cultivar PI 478652's PaAos would identify a high rubber producing guayule; whereas screening for guayule with PaAos with a higher level of functionality would identify a low rubber producing guayule.
Because changes in PaAos functionality changes the amount of rubber produced by guayule, this invention also involves increasing a guayule's rubber production by reducing the amount of functional PaAos present in the genetically altered guayule (compared to the amount of functional PaAos present in non-altered guayule). In one embodiment, the genetically altered guayule has a mutation in PaAos, such as, a null mutation which results in (i) no protein is produced, (ii) a truncated protein is produced which has no functionality, or (iii) a full-length protein is produced which has no functionality. Other mutations simply reduce the functionality (activity) of PaAos. Non-limiting examples of mutations that reduce or eliminate PaAos functionality include (i) changing a codon encoding an amino acid to a stop codon (see Table 1 supra for the sequence of stop codons), (ii) changing the translation initiation codon (ATG) to any other codon to disrupt protein translation, (iii) changing a ribosome binding site's sequence to disrupt protein translation, (iv) changing one or more splice site codons to alter protein sequence, (v) deleting some or all of the gene's DNA sequence, (vi) inserting DNA into the gene, and (vii) changing one or more DNA codon sequences to encode non-conservative amino acids. Within SEQ ID NO: 9, nucleotides 34-36 encode ATG, the translation initiation codon for G7-11 guayule. Similarly, nucleotides 1-3 of SEQ ID NOs: 12 and 14 encode ATG, the translation initiation codon for cultivars PI 478652 and W6549, respectfully. Thus, a change in the nucleotide sequence of the equivalent codon in any other guayule would have the same result. Within PaAos, non-conservative amino acid substitutions at D318, S332, E336, R339, S359, I408, S411, and/or L459 (to name a few) result in PaAos with reduced functionality. One alters guayule DNA using the methods described herein and assesses changes in PaAos functionality via the methods described herein (e.g., assessing the amount of rubber produced by the altered guayule) or using methods known to one of ordinary skill in the art. One can utilize SNPs, antibodies, and other methods to identify the guayule that encode the altered amino acids. When no functional PaAos is produced or when PaAos with reduced functionality is produced, then it is understood that the altered guayule produces “a reduced amount of functional PaAos”. Such altered guayule producing a reduced amount of functional PaAos is another embodiment of this invention.
Because this invention involves production of genetically altered plants and involves recombinant DNA techniques, the following definitions are provided to assist in describing this invention. The terms “isolated”, “purified”, or “biologically pure” as used herein, refer to material that is substantially or essentially free from components that normally accompany the material in its native state or when the material is produced. In an exemplary embodiment, purity and homogeneity are determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A nucleic acid or particular bacteria that are the predominant species present in a preparation is substantially purified. In an exemplary embodiment, the term “purified” denotes that a nucleic acid or protein that gives rise to essentially one band in an electrophoretic gel. Typically, isolated nucleic acids or proteins have a level of purity expressed as a range. The lower end of the range of purity for the component is about 60%, about 70% or about 80% and the upper end of the range of purity is about 70%, about 80%, about 90% or more than about 90%.
The term “gene” refers to a DNA sequence involved in producing a RNA or polypeptide or precursor thereof. The polypeptide or RNA is encoded by a full-length coding sequence (cds) or by intron-interrupted portions of the coding sequence, such as exon sequences. In one embodiment of this invention, the gene involved is Parthenium argentatum allene oxide synthase (PaAos or Aos). PaAos cDNA and amino acid sequence is found in GenBank accession number X78166.2 which is cultivar G7-11 (wild-type/non-altered) (USDA publicly released guayule Germplasm line AZ-2 (Reg. No. GP-9; PI 599676)). The cDNA sequence is in SEQ ID NO: 9; the protein sequence is in SEQ ID NO: 10. SEQ ID NO: 12 is the cDNA sequence for PaAos and SEQ ID NO: 13 is the amino acid sequence for PaAos in guayule W6549 cultivar. SEQ ID NO: 14 is the cDNA sequence for PaAos and SEQ ID NO: 15 is the amino acid sequence for PaAos in guayule 478652 cultivar. A molecular marker uses SNPs within PaAos to differentiate the cultivars with differences in PaAos amino acid sequences which result in PaAos with different functionalities.
The term “nucleic acid” as used herein, refers to a polymer of ribonucleotides or deoxyribonucleotides. Typically, “nucleic acid” polymers occur in either single- or double-stranded form, but are also known to form structures comprising three or more strands. The term “nucleic acid” includes naturally occurring nucleic acid polymers as well as nucleic acids comprising known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Exemplary analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). “DNA”, “RNA”, “polynucleotides”, “polynucleotide sequence”, “oligonucleotide”, “nucleotide”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, “nucleic acid fragment”, and “isolated nucleic acid fragment” are used interchangeably herein.
For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp), or nucleotides (nt). Estimates are typically derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kiloDaltons (kDa) or amino acid residue numbers. Proteins sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from published protein sequences.
Unless otherwise indicated, a particular nucleic acid sequence for each amino acid substitution (alteration) also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), the complementary (or complement) sequence, and the reverse complement sequence, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98(1994)). Because of the degeneracy of nucleic acid codons, one can use various different polynucleotides to encode identical polypeptides. Table 1, infra, contains information about which nucleic acid codons encode which amino acids and is useful for determining the possible nucleotide substitutions that are included in this invention.

TABLE 1

Amino
acid	Nucleic acid codons

Ala/A	GCT, GCC, GCA, GCG

Arg/R	CGT, CGC, CGA, CGG, AGA, AGG

Asn/N	AAT, AAC

Asp/D	GAT, GAC

Cys/C	TGT, TGC

Gln/Q	CAA, CAG

Glu/E	GAA, GAG

Gly/G	GGT, GGC, GGA, GGG

His/H	CAT, CAC

Ile/I	ATT, ATC, ATA

Leu/L	TTA, TTG, CTT, CTC, CTA, CTG

Lys/K	AAA, AAG

Met/M	ATG

Phe/F	TTT, TTC

Pro/P	CCT, CCC, CCA, CCG

Ser/S	TCT, TCC, TCA, TCG, AGT, AGC

Thr/T	ACT, ACC, ACA, ACG

Trp/W	TGG

Tyr/Y	TAT, TAC

Val/V	GTT, GTC, GTA, GTG

Stop	TAA, TGA, TAG

In addition to the degenerate nature of the nucleotide codons which encode amino acids, alterations in a polynucleotide that result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. “Conservative amino acid substitutions” are those substitutions that are predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference protein. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine or histidine, is also expected to produce a functionally equivalent protein or polypeptide. Table 2 provides a list of exemplary conservative amino acid substitutions. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain. In another embodiment, groups of amino acids that are conservative substitutions for each other are (i) alanine (Ala or A), serine (Ser or S), and threonine (Thr or T); (ii) aspartic acid (Asp or D) and glutamic acid (Glu or E); (iii) asparagine (Asn or N) and glutamine (Gln or Q); (iv) arginine (Arg or R) and lysine (Lys or K); (v) isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), and valine (Val or V); and (vi) phenylalanine (Phe or F), tyrosine (Tyr or Y), and tryptophan (Trp or W). See, Creighton, Proteins, W.H. Freeman and Co. (1984), contents of which are expressly incorporated herein. In yet another embodiment, amino acid(s) that are conservative substitutes for one amino acid are grouped by the following characteristics: aliphatic amino acids (alanine, glycine, isoleucine, leucine, and valine); hydroxyl or sulfur containing amino acids (cysteine, serine, methionine, and threonine); cyclic (proline); aromatic (phenylalanine, tryptophan, and tyrosine); basic (arginine, histidine, and lysine); acidic (aspartate and glutamate); and uncharged (asparagine and glutamine). As discussed below, wild-type guayule can be “high” rubber producers or “low” rubber producers. In both types of guayule, there are several amino acid changes in PaAos that be used to distinguish the “high” and “low” rubber producers. As such, one may change DNA encoding one, two, or more of the amino acids to change a “low” rubber producing guayule into a “high” rubber producing guayule. Alternatively, one may change the DNA to encode an amino acid that is a conservative substitute (per Table 2) of the one, two, or more amino acids different in the “high” rubber producing guayule. Further, because the “high” rubber producing guayule discussed below have several amino acids different in PaAos than the “low” rubber producing guayule, the invention also includes any conservative amino acids changes that can be made in these one, two, or more amino acids.

	TABLE 2

	Amino Acid	Conservative Substitute

	Ala	Gly, Ser
	Arg	His, Lys
	Asn	Asp, Gln, His
	Asp	Asn, Glu
	Cys	Ala, Ser
	Gln	Asn, Glu, His
	Glu	Asp, Gln, His
	Gly	Ala
	His	Asn, Arg, Gln, Glu
	Ile	Leu, Val
	Leu	Ile, Val
	Lys	Arg, Gln, Glu
	Met	Ile, Leu
	Phe	His, Leu, Met, Trp, Tyr
	Ser	Cys, Thr
	Thr	Ser, Val
	Trp	Phe, Tyr
	Tyr	His, Phe, Trp
	Val	Ile, Leu, Thr

The term “primer” refers to an oligonucleotide which may act as a point of initiation of DNA extension. A primer may occur naturally, as in a purified restriction digest, or may be produced synthetically.
A primer is selected to be “substantially complementary” to a strand of specific sequence of the template. A primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence is sufficiently complementary with the sequence of the template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer.
Oligonucleotides and polynucleotides that are not commercially available can be chemically synthesized e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), or using an automated synthesizer, as described in Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984). Other methods for synthesizing oligonucleotides and polynucleotides are known in the art. Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).
The terms “identical” or percent “identity”, in the context of two or more polynucleotides or polypeptide sequences, refer to two or more sequences or sub-sequences that are the same or have a specified percentage of nucleotides or amino acids (respectively) that are the same (e.g., 80%, 85% identity, 90% identity, 99%, or 100% identity), when compared and aligned for maximum correspondence over a designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.
The phrase “high percent identical” or “high percent identity”, in the context of two polynucleotides or polypeptides, refers to two or more sequences or sub-sequences that have at least about 80%, identity, at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide or amino acid identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In an exemplary embodiment, a high percent identity exists over a region of the sequences that is at least about 16 nucleotides or amino acids in length. In another exemplary embodiment, a high percent identity exists over a region of the sequences that is at least about 50 nucleotides or amino acids in length. In still another exemplary embodiment, a high percent identity exists over a region of the sequences that is at least about 100 nucleotides or amino acids or more in length. In one exemplary embodiment, the sequences are high percent identical over the entire length of the polynucleotide or polypeptide sequences.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, or alternative parameters designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison is conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of various algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and/or by manual alignment and visual inspection (see, e.g., Ausubel et al. (eds.), Current Protocols in Molecular Biology, 1995 supplement).
The “complement” of a particular polynucleotide sequence is that nucleotide sequence which would be capable of forming a double-stranded DNA or RNA molecule with the represented nucleotide sequence, and which is derived from the represented nucleotide sequence by replacing the nucleotides by their complementary nucleotide according to Chargaff's rules (A< >T; G< >C) and reading in the 5′ to 3′ direction, i.e., in opposite direction of the represented nucleotide sequence (reverse complement).
In one embodiment of the invention, sense and antisense RNAs and dsRNA can be separately expressed in-vitro or in-vivo. In-vivo production of sense and antisense RNAs may use different chimeric polynucleotide constructs using the same or different promoters or using an expression vector containing two convergent promoters in opposite orientation. The sense and antisense RNAs which are formed (e.g., in the same host cells or synthesized) then combine to form dsRNA. To be clear, whenever reference is made herein to a dsRNA chimeric or fusion polynucleotide or a dsRNA molecule, that such dsRNA formed (e.g., in plant cells) from sense and antisense RNA produced separately is also included. Also, synthetically made dsRNA and self-annealing RNA strands are included herein when the sense and antisense strands are present together.
As used herein, the term “promoter” refers to a polynucleotide that, in its native state, is located upstream or 5′ to a translational start codon of an open reading frame (or protein-coding region) and that is involved in recognition and binding of RNA polymerase and other proteins (trans-acting transcription factors) to initiate transcription. A “plant promoter” is a native or non-native promoter that is functional in plant cells, even if the promoter is present in a microorganism that infects plants or a microorganism that does not infect plants. The promoters that are predominately functional in a specific tissue or set of tissues are considered “tissue-specific promoters”. A plant promoter can be used as a 5′ regulatory element for modulating expression of a particularly desired polynucleotide (heterologous polynucleotide) operably linked thereto. When operably linked to a transcribable polynucleotide, a promoter typically causes the transcribable polynucleotide to be transcribed in a manner that is similar to that of which the promoter is normally associated.
Plant promoters include promoters produced through the manipulation of known promoters to produce artificial, chimeric, or hybrid promoters. Such promoters can also combine cis-elements from one or more promoters, for example, by adding a heterologous regulatory element to an active promoter with its own partial or complete regulatory elements. The term “cis-element” refers to a cis-acting transcriptional regulatory element that confers an aspect of the overall control of gene expression. A cis-element may function to bind transcription factors, trans-acting protein factors that regulate transcription. Some cis-elements bind more than one transcription factor, and transcription factors may interact with different affinities with more than one cis-element.
The term “vector” refers to DNA, RNA, a protein, or polypeptide that are to be introduced into a host cell or organism. The polynucleotides, protein, and polypeptide which are to be introduced into a host may be therapeutic or prophylactic in nature; may encode or be an antigen; may be regulatory in nature; etc. There are various types of vectors including viruses, viroids, plasmids, bacteriophages, cosmids, and bacteria.
An expression vector is nucleic acid capable of replicating in a selected host cell or organism. An expression vector can replicate as an autonomous structure, or alternatively integrate, in whole or in part, into the host cell chromosomes or the nucleic acids of an organelle, or it is used as a shuttle for delivering foreign DNA to cells, and thus replicate along with the host cell genome. Thus, an expression vector are polynucleotides capable of replicating in a selected host cell, organelle, or organism, e.g., a plasmid, virus, artificial chromosome, nucleic acid fragment, and for which certain genes on the expression vector (including genes of interest) are transcribed and translated into a polypeptide or protein within the cell, organelle or organism; or any suitable construct known in the art, which comprises an “expression cassette”. In contrast, as described in the examples herein, a “cassette” is a polynucleotide containing a section of an expression vector. The use of the cassettes assists in the assembly of the expression vectors. An expression vector is a replicon, such as plasmid, phage, virus, chimeric virus, or cosmid, and which contains the desired polynucleotide sequence operably linked to the expression control sequence(s).
A heterologous polynucleotide sequence is operably linked to one or more transcription regulatory elements (e.g., promoter, terminator and, optionally, enhancer) such that the transcription regulatory elements control and regulate the transcription and/or translation of that heterologous polynucleotide sequence. A cassette has the heterologous polynucleotide operably linked to one or more transcription regulatory elements. As used herein, the term “operably linked” refers to a first polynucleotide, such as a promoter, connected with a second transcribable polynucleotide, such as a gene of interest, where the polynucleotides are arranged such that the first polynucleotide affects the transcription of the second polynucleotide. In some embodiments, the two polynucleotide molecules are part of a single contiguous polynucleotide. In other embodiments, the two polynucleotides are adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell. Similarly, a terminator is operably linked to the polynucleotide of interest if the terminator regulates or mediates transcription of the polynucleotide of interest, and in particular, the termination of transcription. Constructs of the present invention would typically contain a promoter operably linked to a transcribable polynucleotide operably linked to a terminator.
The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, organism, nucleic acid, protein or vector, has been altered by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so altered. Thus, for example, recombinant cells may express genes/polynucleotides that are not found within the native (non-recombinant or non-altered or wild-type) form of the cell or express native genes in an otherwise abnormal amount—over-expressed, under-expressed or not expressed at all—compared to the non-altered cell or organism. In particular, one alters the genomic DNA of a non-altered plant by molecular biology techniques that are well-known to one of ordinary skill in the art and generate a recombinant plant.
The terms “transgenic”, “transformed”, “transformation”, and “transfection” are similar in meaning to “recombinant” “Transformation”, “transgenic”, and “transfection” refer to the transfer of a polynucleotide into a host organism or into a cell. Such a transfer of polynucleotides may result in genetically stable inheritance of the polynucleotides or in the polynucleotides remaining extra-chromosomally (not integrated into the chromosome of the cell). Genetically stable inheritance may potentially require the transgenic organism or cell to be subjected for a period of time to one or more conditions which require the transcription of some or all of the transferred polynucleotide in order for the transgenic organism or cell to live and/or grow. Polynucleotides that are transformed into a cell but are not integrated into the host's chromosome remain as an expression vector within the cell. One may need to grow the cell under certain growth or environmental conditions in order for the expression vector to remain in the cell or the cell's progeny. Further, for expression to occur the organism or cell may need to be kept under certain conditions. Genetically altered organisms or cells containing the recombinant polynucleotide are referred to as “transgenic” or “transformed” organisms or cells or simply as “transformants”, as well as recombinant organisms or cells.
A genetically altered organism is any organism with any changes to its genetic material involving the invention described herein, whether in the nucleus or cytoplasm (organelle). As such, a genetically altered organism may be a recombinant or transformed organism. A genetically altered organism may also be an organism that was subjected to one or more mutagens or the progeny of an organism that was subjected to one or more mutagens and has mutations in its DNA caused by the one or more mutagens, as compared to the wild-type organism (i.e., organism not subjected to the mutagens) or the non-altered organism (i.e., one that contains alterations that are not the subject matter of this invention). Also, an organism that has been bred to incorporate a mutation into its genetic material is a genetically altered organism.
The term “altered” means that a change occurred compared to the “non-altered” item. However, a “non-altered” item could contain changes that are induced by man, but those changes are not the subject matter of the inventions described herein. For example, a non-altered guayule contains none of the described genetic changes nor has been treated with any of the described external substance, but may contain pre-existing changes which are not part of this invention. An altered guayule (which also is a genetically altered guayule) may contain DNA mutations which change PaAos' amino acid sequence, even if that sequence exists in a non-altered plant. Such DNA mutations may be induced by a mutagen (EMS, UV light, other radiation, etc.).
Transformation and generation of genetically altered monocotyledonous and dicotyledonous plant cells is well known in the art. See, e.g., Weising, et al., Ann. Rev. Genet. 22:421-477 (1988); U.S. Pat. No. 5,679,558; Agrobacterium Protocols, ed: Gartland, Humana Press Inc. (1995); and Wang, et al. Acta Hort. 461:401-408 (1998). A method to generate genetically altered guayule is described in U.S. Pat. No. 9,018,449 (Dong & Cornish) and in Dong, et al., Plant Cell Reports 25:26-34 (2006). A method to generate transplastomic guayule is provided in U.S. Patent Application Publication 2014/0325699, contents of which are expressly incorporated herein. The choice of method varies with the type of plant to be transformed, the particular application and/or the desired result. The appropriate transformation technique is readily chosen by the skilled practitioner.
A polynucleotide encoding PaAos (SEQ ID NOs: 9, 12, and/or 14), the reverse complement of PaAos, or a portion thereof (e.g., SEQ ID NO: 11), operably linked to one or two appropriate promoters, can be stably inserted in a conventional manner into the genome (cytoplasmic genome or nucleic genome) of a single plant cell, and the altered plant cell can be used in a conventional manner to produce a genetically altered plant that produces the dsRNA of this invention. In this regard, a disarmed Ti-plasmid, containing the polynucleotide of this invention, in Agrobacterium tumefaciens can be used to genetically alter the plant cell, and thereafter, a genetically altered plant can be regenerated from the genetically altered plant cell using the procedures described in the art, for example, in EP 0 116 718, EP 0 270 822, WO 84/02913 and EP 0 242 246. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, in Handbook of Plant Cell Culture, pp. 124-176, MacMillan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, in Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee, et al., Ann. Rev. of Plant Phys. 38:467-486 (1987).
Preferred Ti-plasmid vectors each contain the polynucleotides described herein between the border sequences, or at least located to the left of the right border sequence, of the T-DNA of the Ti-plasmid. Of course, other types of vectors may be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example in EP 0 233 247), pollen mediated transformation (as described, for example in EP 0 270 356, WO 85/01856, and U.S. Pat. No. 4,684,611), plant RNA virus-mediated transformation (as described, for example in EP 0 067 553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation (as described, for example in U.S. Pat. No. 4,536,475), and other methods such as the methods for transforming certain lines of corn (e.g., U.S. Pat. No. 6,140,553; Fromm, et al., Bio/Technology 8:833-839 (1990); Gordon-Kamm, et al., The Plant Cell 2:603-618 (1990) and rice (Shimamoto, et al., Nature 338:274-276 (1989); Datta et al., Bio/Technology 8:736-740 (1990)) and the method for transforming monocots generally (WO 92/09696). For cotton transformation, the method described in WO 2000/71733 can be used. For soybean transformation, reference is made to methods known in the art, e.g., Hinchee, et al. (Bio/Technology 6:915 (1988)) and Christou, et al. (Trends Biotechnology 8:145 (1990)) or the method of WO 00/42207.
The resulting genetically altered plant can be used in a conventional plant breeding scheme to produce more genetically altered plants with the same characteristics or to introduce the polynucleotide into other varieties of the same or related plant species. Seeds, which are obtained from the genetically altered plants, contain the expression vector as a stable genomic insert. Altered plants include plants having or derived from root stocks of plants containing the expression vector. Hence, any non-altered grafted plant parts inserted on a genetically altered plant or plant part are included in the invention.
For a genetically altered plant that produces dsRNA, one constructs an expression vector or cassette (made from DNA) that encodes, at a minimum, a first promoter and the dsRNA sequence of interest such that the promoter sequence is 5′ (upstream) to and operably linked to the dsRNA sequence. The expression vector or cassette may optionally contain a second promoter (same as or different from the first promoter) upstream and operably linked to the reverse complementary sequence of the dsRNA sequence such that two strands of RNA that are complementary to each other are produced. Alternatively, the expression vector or cassette can contain one promoter operably linked to both the dsRNA sequence (sense strand) in question and the complement or reverse complement of the dsRNA sequence (anti-sense strand) in question, such that the transcribed RNA bends on itself and the two desires sequences anneal. Alternatively, a second expression vector or cassette (made from DNA) may encode, at a minimum, a second promoter (same as or different from the promoter) operably linked to the reverse complementary sequence of the dsRNA such that two strands of complementary RNA are produced in the plant. The expression vector(s) or cassette(s) is/are inserted in a plant cell genome (nuclear or cytoplasmic). The promoter(s) used should be a promoter(s) that is/are active in a plant and is/are heterologous to PaAos (not normally driving the transcription of RNA of genomic PaAos). Of course, the expression vector or cassette may have other transcription regulatory elements, such as enhancers, terminators, etc.
Promoters (and more specifically, heterologous promoters for PaAos) that are active in plants are well-known in the field. Such promoters may be constitutive, inducible, and/or tissue-specific. Non-limiting examples of constitutive plant promoters include 35S promoters of the cauliflower mosaic virus (CaMV) (e.g., of isolates CM 1841 (Gardner, et al., Nucleic Acids Research 9:2871-2887 (1981)), CabbB-S (Franck, et al., Cell 21:285-294 (1980)) and CabbB-JI (Hull and Howell, Virology 86:482-493 (1987))), ubiquitin promoter (e.g., the maize ubiquitin promoter of Christensen, et al., Plant Mol. Biol. 18:675-689 (1992)), gos2 promoter (de Pater, et al., The Plant J. 2:834-844 (1992)), emu promoter (Last, et al., Theor. Appl. Genet. 81:581-588 (1990)), actin promoter (see, e.g., An, et al., The Plant J. 10:107 (1996)) and Zhang, et al., The Plant Cell 3:1155-1165 (1991)); Cassava vein mosaic virus promoters (see, e.g., WO 97/48819 and Verdaguer, et al., Plant Mol. Biol. 37:1055-1067 (1998)), the pPLEX series of promoters from Subterranean Clover Stunt Virus (WO 96/06932, particularly the S4 or S7 promoter), alcohol dehydrogenase promoter (e.g., pAdh1S (GenBank accession numbers X04049, X00581)), and the TR1′ promoter and the TR2′ promoter which drive the expression of the 1′ and 2′ genes, respectively, of the T-DNA (Velten, et al., EMBO J. 3:2723-2730 (1984)). Tissue-specific promoters are promoters that direct a greater level of transcriptional expression in some cells or tissues of the plant than in other cells or tissue. Non-limiting examples of tissue-specific promoters include the phosphoenolpyruvate carboxylase (PEP or PPC1) promoter (Pathirana, et al., Plant J. 12:293-304 (1997), and Kausch, et al., Plant Mol. Biol. 45(1):1-15 (2001)), chlorophyll A/B binding protein (CAB) promoter (Bansal, et al., Proc. Natl. Acad. Sci. USA 89(8):3654-8 (1992)), small subunit of ribulose-1,5-bisphosphate carboxylase (ssRBCS) promoter (Bansal, et al., Proc. Natl. Acad. Sci. USA 89(8):3654-8 (1992)), senescence activated promoter (SEE1) (Robson, et al., Plant Biotechnol. J. 2(2):101-12 (2004)), and sorghum leaf primoridia specific promoter (RS2) (GenBank Accession No. E1979305.1). These promoters (PPC1, CAB, ssRBCS, SSE1, and RS2) are all active in the aerial part of a plant. Further, the PPC1 promoter is a strong promoter for expression in vascular tissue. Some examples of phloem specific promoters are the sucrose synthase-1 promoters (CsSUS1p and CsSUS1p-2) (Singer et al., Planta 234:623-637 (2011)) and the phloem protein-2 promoter (CsPP2) (Miyata et al., Plant Cell Report 31(11):2005-2013 (2012)) from Citrus sinensis. Alternatively, a plant-expressible promoter may also be a wound-inducible promoter, such as the promoter of the pea cell wall invertase gene (Zhang, et al., Plant Physiol. 112:1111-1117 (1996)).
Other types of RNA polymerase promoters that may be used are promoters from microorganisms, such as, but not limited to the bacteriophage T7 RNA polymerase promoter, yeast Galactose (GAL1) promoter, yeast glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, yeast Alcohol Oxidase (AOX) promoter.
Other elements used to increase transcription expression in plant cells include, but are not limited to, an intron (e.g., hsp70 intron) at the 5′ end or 3′ end of the chimeric gene, or in the coding sequence of the chimeric dsRNA gene (such as, between the region encoding the sense and antisense portion of the dsRNA), promoter enhancer elements, duplicated or triplicated promoter regions, 5′ leader sequences different from the chimeric gene or different from an endogenous (plant host) gene leader sequence, 3′ untranslated sequences different from the chimeric gene or different from an endogenous (plant host) 3′ untranslated sequence.
The expression vector or cassette could contain suitable 3′ untranslated transcription regulation sequences (i.e., transcript formation and polyadenylation sequences). Potential polyadenylation and transcript formation sequences include those sequences in the nopaline synthase gene (Depicker, et al., J. Molec. Appl. Genetics 1:561-573 (1982)), the octopine synthase gene (Gielen, et al., EMBO J. 3:835-845 (1984)), the SCSV or the Malic enzyme terminators (Schunmann, et al., Plant Functional Biology 30:453-460 (2003)), and the T-DNA gene 7 (Velten and Schell, Nucleic Acids Research 13:6981-6998 (1985)).
The term “plant” includes whole plants, plant organs, progeny of whole plants or plant organs, embryos, somatic embryos, embryo-like structures, protocorms, protocorm-like bodies (PLBs), and suspensions of plant cells. Plant organs comprise, e.g., shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, trichomes and the like). The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to the molecular biology and plant breeding techniques described herein, specifically angiosperms (monocotyledonous (monocots) and dicotyledonous (dicots) plants). It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous. The genetically altered plants described herein are guayule plants.
Rubber yield may be expressed as a product of rubber content (% rubber) and biomass (dry weight/unit area). Thus, rubber yield may be improved by increasing either biomass and/or rubber content. The altered guayule described herein produce more rubber and have higher rubber content than non-altered guayule, thereby increasing the processing efficiency of the guayule shrub.
Various methods exist to create a mutation. These methods are well-known to one of ordinary skill in the art. One method is by transforming the plant with a plasmid containing 5′ sequence and 3′ sequence of the gene and allowing a cross-over event to occur, thereby excising the DNA from the plant's genome that is between the plasmid's 5′ sequence and 3′ sequence. Also, one can use transposon-mediated mutation to delete or add DNA to PaAos which would result in the encoded protein having a reduced functionality compared to a non-altered PaAos. Two other methods involve using a chemical mutagen (such as ethyl methanesulfonate (EMS)) or physical agents (radiation, UV, or proton, for example) to generate genetic mutations in plant cells and/or germplasm. Also, one may use TALEN or CRISPR-Cas9 to mutate the sequence of the target gene (PaAos) such that a desired mutation is generated. One of ordinary skill in the art can also use targeted cleavage events to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination and integration at predetermined chromosomal locations to generate one or more of the null mutations discussed above or to reduce the mutated protein's functionality. Nucleotide editing techniques are well-known and described in Urnov, et al., Nature 435(7042):646-51 (2010); U.S. Patent Publications 2003/0232410, 2005/0208489, 2005/0026157, 2005/0064474, 2006/0188987, 2009/0263900, 2009/0117617, 2010/0047805, 2011/0207221, 2011/0301073, 2011/089775, 2011/0239315, and 2011/0145940; and International Publication WO 2007/014275, the disclosures of which are incorporated by reference in their entireties for all purposes. Cleavage occurs by using specific nucleases such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TALENs), or using the CRISPR/Cas9 system with an engineered crRNA/tracr RNA (‘single guide RNA’) to guide specific cleavage. U.S. Patent Publication 2008/0182332 describes the use of non-canonical zinc finger nucleases (ZFNs) for targeted modification of plant genomes; U.S. Patent Publication 2009/0205083 describes ZFN-mediated targeted modification of a plant EPSPS locus; U.S. Patent Publication 2010/0199389 describes targeted modification of a plant Zp15 locus and U.S. Patent Publication No. 20110167521 describes targeted modification of plant genes involved in fatty acid biosynthesis. In addition, Moehle, et al., Proc. Natl. Acad. Sci. USA 104(9):3055-3060 (2007) describes using designed ZFNs for targeted gene addition at a specified locus. U.S. Patent Publication 2011/0041195 describes methods of making homozygous diploid organisms. Information on CRISPR/Cas9 system is found, e.g., at en.wikipedia.org/wiki/CRISPR; neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology; and Cong, et al., Science, 339:819-823 (2013). Sigma-Aldrich (St. Louis, Mo.) and Origene Technologies, Inc. (Rockville, Md.) are among the companies that sell CRISPR/Cas9 kits.
After using any of these various methods to induce genetic alterations in a cell's genome, one induces the treated cells to grow into plants and then screen the plants using the methods described herein for PaAos having reduced or no functionality, and/or for reduced amounts of PaAos or no PaAos (via reduction in gene expression and/or mRNA translation and/or other mechanism), and/or for elevated production of rubber (compared to amounts present in non-altered plants). Thus, another embodiment of this invention is the generation of altered guayule having a genetic alteration in PaAos such that the altered guayule produces more rubber than produced by non-altered guayule.
In another embodiment, one synergistically increases the amount of rubber produced by exposing an altered guayule to cold temperatures (between approximately 7° C. and approximately 15° C. or between approximately 10° C. and approximately 15° C.; approximately 8 hours per day) for approximately two weeks or more. The altered guayule may contain one of more of the following alterations: (1) DNA encoding (i) anti-sense RNA for PaAos, (ii) double-stranded RNA for PaAos, (iii) a mutation within PaAos that encodes a PaAos with reduced or no function; and/or (2) exogenously administered PaAos dsRNA. The combination of any of the above alterations and exposure to cold temperatures (between approximately 7° C. and approximately 15° C. or between approximately 10° C. and approximately 15° C.; approximately 8 hours per day) for approximately two weeks or more result in production of increased amounts of rubber than produced by the non-altered plant exposed to the same temperatures for the same time period.
Many techniques involving molecular biology discussed herein are well-known to one of ordinary skill in the art and are described in, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual 4th ed. 2012, Cold Spring Harbor Laboratory; Ausubel et al. (eds.), Current Protocols in Molecular Biology, 1994—current, John Wiley & Sons; and Kriegler, Gene Transfer and Expression: A Laboratory Manual (1993). Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology maybe found in e.g., Benjamin Lewin, Genes IX, Oxford University Press, 2007 (ISBN 0763740632); Krebs, et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
The terms “approximately” and “about” refer to a quantity, level, value or amount that varies by as much as 30% in one embodiment, or in another embodiment by as much as 20%, and in a third embodiment by as much as 10% to a reference quantity, level, value or amount. As used herein, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a bacterium” includes both a single bacterium and a plurality of bacteria.
The term “nucleic acid consisting essentially of”, “polynucleotide consisting essentially of”, and “RNA consisting essentially of”, and grammatical variations thereof, means a polynucleotide that differs from a reference nucleic acid sequence by 20 or fewer nucleotides and also perform the function of the reference polynucleotide sequence. Such variants include sequences which are shorter or longer than the reference nucleic acid sequence, have different residues at particular positions, or a combination thereof.
Having now generally described this invention, the same will be better understood by reference to certain specific examples and the accompanying drawings, which are included herein only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims. The examples and drawings describe at least one, but not all embodiments, of the inventions claimed. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

Example 1. Construction of Genetically Altered Guayule

To better understand the role of PaAos in rubber synthesis, genetically altered P. argentatum plants are generated in which either PaAos is over-expressed or PaAos is silenced by RNAi. The various plasmids used to achieve the overexpression or silencing of PaAos in guayule are shown in FIG. 1. To generate these plasmids, the guayule Aos (PaAos) is amplified by PCR using genomic DNA as a template. The primers used to amplify PaAos are designed from the cDNA PaAos sequence published in NCBI database (GeneBank accession no. X78166.2) and have the following sequences: forward primer 5′-cttaagaggtggtATGGACCCATCGTCTAAACCC-3′ (SEQ ID NO: 1) and reverse primer 5′-ggatccTCATATACTAGCTCTCTTCAGGG-3′ (SEQ ID NO: 2). The nucleotides in lower case and underlined in the forward primer are the recognition nucleotides for restriction enzyme AflII; the nucleotides in lower case and underlined in the reverse primer are the recognition nucleotides for restriction enzyme BamHI. The PCR cycle program is the following: 94° C. for 2 minutes (initial heating step) and PaAos is amplified at 40 cycles of 94° C. for 30 seconds (denaturation), 71° C. for 30 seconds (annealing) and 68° C. for 1 minute (extension) and an additional 5 minutes extension at 68° C. The resulting amplicon is purified and subcloned into pGEM T Easy vector (Promega, Madison, Wis.) using manufacturer's recommended protocol and sequenced to confirm the sequence of the plasmids. The cDNA sequence is in SEQ ID NO: 9 and the amino acid sequence is in SEQ ID NO: 10. Subsequently, the PaAos amplicon is cut using AflII and BamHI. Plasmid pND6 has a Nos promoter driving the NPTII gene for conferring kanamycin resistance and a potato ubiquitin promoter (Garbarino and Belknap, Plant Mol. Biol. 24:119-127 (1994)) controlling the GUSplus gene (CambiaLabs, Canberra, Australia). See FIG. 1. Plasmid pND6-Aos (FIG. 1) is generated by replacing the GUSplus gene in pND6 with cDNA PaAos sequence (SEQ ID NO: 9). Plasmid pND6-AosiL (FIG. 1) is generated by replacing the GUSplus gene in pND6 with an inverted repeat of a partial cDNA PaAos sequence (SEQ ID NO: 11) containing a loop sequence of the Bar gene between the inverted repeat of the partial cDNA PaAos sequence; the complete sequence replacing GUSplus is SEQ ID NO: 26. The plasmids pND6, pND6-AosiL, and pND6-Aos are used to transform Agrobacterium EHA101 competent cells using the protocol described in Hood, et al., J. Bacteriol. 168:1291-1301 (1986).
The transformed Agrobacterium EHA101 either harboring pND6, pND6-AosiL, or pND6-Aos are used to transform guayule G7-11 using the protocols set forth below. See also Dong, et al. (2006) and Dong, et al., Industrial Crops and Products 46:15-24 (2013). For Agrobacterium transformation, the overnight Agrobacterium culture are prepared by inoculating 50 μL glycerol stock into a 50 mL Falcon tube containing 5 mL LB medium plus 40 mg/L rifampcin and 200 mg/L spectinomycin, and shaking at 200 rpm at 28° C. The suspension then is centrifuged for 15 minutes at 1600×g at room temperature. The supernatant is discarded, and the pellet is re-suspended in 25 mL of inoculation solution ( 1/10 MS salts plus BA (2 mg/L), NAA (0.5 mg/L), glucose (10 g), acetosyringone (200 μM), pluronic F68 (0.05%), pH=5.2 (PhytoTechnology Labs, Shawnee Mission, Kans.)). For leaf transformation, leaf sections are cut from the plants in the Magenta boxes (Caisson Labs, Smithfield, Utah). The adaxial side of each leaf is placed facing up in a Petri dish containing 5 ml Agrobacterium suspension. The leaf is cut into ˜10 mm strips and immediately placed in an empty Petri dish in non-overlapping manner. When this Petri dish is full, all leaf strips are blotted with the filter paper and placed into another empty Petri dish. The Petri dish is sealed by parafilm and left in the dark at room temperature. The co-cultivation is replaced by this co-desiccation according to Cheng, et al., In Vitro Cell Dev. Biol. Plant, 39, 595-604 (2003). After three days, leaf strips are transferred to MSB1T (MS medium with BA (1 mg/L), sucrose (30 g/L), phytagel (3 g/L), and timentin (400 mg/L)) (Cheng, et al., Plant Cell Rep., 17(8):646-649 (1998)) for recovery at low light for 5 days. The leaf strips are then transferred to MSB1TK30 (MS medium containing BA (1 mg/L), sucrose (30 g/L), phytagel (3 g/L), timentin (250 mg/L), and kanamycin (30 mg/L)) for selection under low light for two weeks. The leaf strips are then subcultured every 2 weeks under high light till green shoots emerged. Green shoots 10 mm and longer are transferred to ½MS10.1TK10 for rooting (same as ½MSI0 but with timentin (250 mg/L) and kanamycin (10 mg/L)). After 2-4 weeks, the rooted plantlets are micropropogated and subsequently transplanted into soil.
While the genetically altered guayule are still growing in tissue culture under selection, the genetically altered plants are screened for integration of the expression vectors, pND6-Aos (PaAos in forward orientation; SEQ ID NO: 9), pND6-AosiL (PaAos in the reverse orientation (a portion of reverse complement of PaAos is SEQ ID NO: 11)), and pND6 (negative control). DNA is extracted from genetically altered plants using Sigma Kit (Sigma-Aldrich, St. Louis, Mo.). Approximately 150 mg leaf tissue (3 leaf tissues) are cut from the plants grown in tissue-cultured, placed into 2 mL tubes and snapped-frozen in liquid nitrogen. A bead is added to pulverize the tissue into a fine powder at a frequency of 30/s for 1 minute using the mixer mill MM 400 tissue lyser (Verder Scientific, Inc., Newtown, Pa.).
PCR is carried out in 50 μL mixture containing Taq 2× Master Mix (New England Biolabs, Ipswich, Mass.), 200 ng guayule genomic DNA or 20 pg plasmid DNA, and 100 ng of PaAos specific primers; namely SEQ ID NOs: 1 and 2 for guayule transformed with pND6-Aos; and SEQ ID NOs: 3 and 4 for guayule transformed with pND6-AosiL. See FIG. 2. After heating the samples to 94° C. for 2 minutes, the reaction proceeds with 35 cycles of 94° C. for 30 seconds, 71° C. to amplify the PaAos in the overexpression lines (pND6-Aos) for 30 seconds or 56° C. for the PaAos in the RNAi lines (pND6-AosiL) for 30 seconds, and 68° C. for 1 minute. A final elongation step is carried out at 68° C. for 5 minutes. PCR products are separated by electrophoresis on a 1% (w/v) agarose gel. The band for the overexpression lines is at ˜1.4 kbp, as expected; the band for the RNAi lines is at ˜0.5 kbp as expected. The genetically altered guayule plants harboring the empty plasmid (pND6 (negative control)) are confirmed by GUS staining (Karcher, S., ABLE 23:29-42 (2002)). Briefly, plant tissues are placed in a 50 mL tube containing GUS assay solution (1 mM X-Gluc (5-bromo-4-chloro-3-indolyl) B-D-glucuronic acid in 50 mM Na₂HPO₄, pH 7.0 and 0.1% Triton X-100). The reaction is incubated at 37° C. for 1 hour followed by washing for 30 minutes with 70% ethanol to extract the chlorophyll.

Example 2. Determination of RNA Expression Levels in Genetically Altered Plants

Guayule containing intact PaAos (non-altered; G7-11) and genetically altered guayule containing one of the plasmids (pND6, pND6-Aos, or pND6-AosiL) are further screened to determine the RNA level (see Table 3). Leaves from the various genetically modified plants (which are grown in tissue culture) are collected and snap-frozen in liquid nitrogen for RNA extraction. RNA is extracted using TRIzol® according to manufacturer's recommended protocol (Ambion, Pittsburgh, Pa.). RNA concentration is quantified with the NanoDrop ND1000 (ThermoScientific, Wilminton, Del.). RNA cleanup is performed using the RNeasy MinElute Cleanup kit according to manufacturer's recommended protocol (Qiagen Inc., Valencia, Calif.). The RNA is eluted with 30-50 μL of RNase-free water along with on-column DNase1 treatment.
Using the RNA isolated from the leaves of the genetically altered plants, cDNA is generated using iScript cDNA synthesis kit (Bio-Rad, Hercules, Calif.) according to the manufacturer's recommended protocol for semi-quantitative PCR and real-time quantitative PCR (qRT-PCR). An amount of 1 μg of RNA is used in the 20 mL reaction mixture. For qRT-PCR, 2 μL of the diluted cDNA (1:20) is used in a 15 μL reaction mixture. In the qRT-PCR volume, 7.5 mL of iQ SYBR® Green Supermix is used (Bio-Rad, Hercules, Calif.). The qRT-PCR is run using the 7500 Fast Real-Time PCR system (Applied Biosystem, Waltham, Mass.) with the following thermal cycle: 95° C. pre-incubation for 3 minutes; amplification is performed for 40 cycles at 95° C. for 15 seconds and at 60° C. for 30 seconds; the dissociation stage is set for 95° C. for 15 seconds, at 60° C. for 1 minute, and at 95° C. for 15 seconds. Each qRT-PCR run is performed with three independent tissue samples, each sample having two technical replicates. The 18S gene (˜200 bp) is used as an internal control. The primers used for each sequence, PCR reaction conditions, and the expected amplicon size are contained in FIG. 2. Crossing point value, which is the point at which the fluorescence crosses the threshold, and melting curve analyses are noted. The melting curve data are collected for all genes to ensure a single peak, indicating amplification of a specific region by a pair of primers. The relative expression values are calculated using the 2(-Delta C(T)) method (Livak and Schmittgen, Methods, 25:402-408 (2001)). See Table 3 below.

	TABLE 3

	P. argentatum	Average Relative
	Genotypes	Expression of Aos

	G7-11	1.02 ± 0.2
	pND6-10	1.14 ± 0.2
	pND6-12	1.02 ± 0.3
	pND6-29	1.04 ± 0.2
	pND6-32	1.00 ± 0.3
	pND6-33	1.11 ± 0.3
	pND6-35	0.90 ± 0.2
	pND6-41	0.91 ± 0.2
	pND6-AosiL_5-1	0.39 ± 0.1*
	pND6-AosiL_7-2	0.49 ± 0.1*
	pND6-AosiL_8-1	0.53 ± 0.1*
	pND6-AosiL_9-15	0.44 ± 0.1*
	pND6-AosiL_9-16	0.37 ± 0.04*
	pND6-AosiL_12-1	0.55 ± 0.1*
	pND6-AosiL_12-3	0.48 ± 0.1*
	pND6-AosiL_13-2	0.55 ± 0.05*
	pND6-AosiL_15-3	0.36 ± 0.1*
	pND6-AosiL_15-4	0.48 ± 0.2*
	pND6-Aos_4-1	2.15 ± 0.1**
	pND6-Aos_4-2	2.11 ± 0.3**
	pND6-Aos_5-1	2.29 ± 0.4**
	pND6-Aos_7-1	2.40 ± 0.6**
	pND6-Aos_7-3	2.11 ± 0.2**
	pND6-Aos_8-2	2.15 ± 0.2**
	pND6-Aos_10-1	2.44 ± 0.7**
	pND6-Aos_10-2	2.12 ± 0.4**
	pND6-Aos_11-5	2.30 ± 0.3**
	pND6-Aos_14-2	2.23 + 0.1**

	G7-11 = wild-type control;
	pND6 = empty vector (pND6 without Aos);
	pND6-AosiL = PaAos is knocked-down/silenced;
	pND6-Aos = PaAos is over-expressed
	Results are average of three independent plants, each plant having three technical replicates.
	* and ** indicate significant difference in comparison to G7-11 guayule and/or pND6 (controls) at p > 0.05 and 0.005, respectively.

Next, to gain more insight as to where the PaAos is spatially located, the expression pattern of PaAos in various guayule tissues is analyzed using qRT-PCR. Total RNA is extracted from leaves, petiole, stem, root, young flower, mature flower, peduncle, stembark of 8-week-old tissue-cultured genetically altered plants as well as 2-month-old greenhouse grown genetically altered plants using the protocol described above. qRT-PCR is performed as described above on these samples of total RNA. Primers (SEQ ID NOs: 7 and 8 in FIG. 2) are designed to amplify ˜200 bp PCR product in PaAos coding sequence. The expression level for each tissue are compared to the tissue cultured and greenhouse leaf tissues, respectively. The 18S gene (˜200 bp) (forward primer is SEQ ID NO: 5 and reverse primer is SEQ ID NO: 6, described supra and in FIG. 2) is used as an internal control. As shown in Table 4, infra, the largest level of PaAos expression is present in the stem, root and stembark tissues, suggesting that these tissues are sites in which PaAos is functioning.

	TABLE 4

	Growth Conditions:

MS Medium

Greenhouse

	Tissue Source	Relative Expression

Leaf	0.98 ± 0.1	1.04 ± 0.2
Petiole	0.31 ± 0.06	0.41 ± 0.1
Stem	2.27 ± 0.2	3.37 ± 0.4
Root	2.47 ± 0.2	3.74 ± 0.3
Young Flower	no data	1.23 ± 0.4
Mature Flower	no data	0.69 ± 0.2
Peduncle	no data	0.25 ± 0.1
Bark	no data	4.49 ± 1.2

The error bars represent tissues collected from 3 individual plants.

Example 3. Rubber Quantification in Tissue

Rooted plantlets (genetically altered, empty vector transformed (pND6 without PaAos), and wild-type control) from transferred shoot tips are grown on half-strength MS medium (PhytoTechnology Laboratories, Overland Park, Kans.) in Magenta boxes (Caisson Labs, Smithfield, Utah) for 6 weeks. The top part of the plantlets are separated from the medium and lyophilized for 48 hours. The dried tissues are placed in a 50 mL stainless steel grinding jar containing grinding ball, frozen in liquid nitrogen for 5 minutes and finely ground using the Retsch mixer mill MM 400 at a frequency of 30/second for 1 minute (Verder Scientific Inc., Newtown, Pa.). Three hundred milligrams (0.3 g) of pulverized tissues are partitioned with Ottawa sand (Fisher Scientific, Fair Lawn, N.J.) and loaded into 11 mL stainless steel extraction cells (Dionex, Sunnyvale, Calif.). Three sequential extractions are performed using the Accelerated Solvent Extractor (ASE 2000; Dionex, Sunnyvale, Calif.): 1. Acetone: to remove resinous material and the low molecular weight organic solubles; 2. Methanol: to remove chlorophyll and other alcohol-soluble materials; 3. Cyclohexane: to remove rubber. Natural rubber is quantified gravimetrically. The percent (%) rubber is the amount (% dw) of cyclohexane extract from 0.3 g dried tissue. The pND6-AosiL plants have 1.5 to 2 times more rubber than G7-11, pND6 and pND6-Aos in tissue-cultured environment (Table 5). In Table 5, the rubber content is quantified from leaf and stems of the indicated guayule genotypes grown in MS media.

TABLE 5

Rubber content of guayule plant shoots determined by
Accelerated Solvent Extraction

	P. argentatum Genotypes	Average Rubber Content (%)

	G7-11.1	1.01 ± .01
	G7-11.2	1.11 ± .02
	pND6-12	1.13 ± 0.2
	pND6-33	1.10 ± 0.1
	pND6-35	1.04 ± 0.2
	pND6-AosiL_5-1	1.8 ± 0.1*
	pND6-AosiL_7-2	2.0 ± 0.3**
	pND6-AosiL_8-1	2.1 ± 0.04***
	pND6-AosiL_8-2	1.7 ± 0.1**
	pND6-AosiL_9-15	1.7 ± 0.02**
	pND6-AosiL_9-16	2.3 ± 0.4*
	pND6-AosiL_12-1	2.46 ± 0.3*
	pND6-AosiL_12-3	1.62 ± 0.002***
	pND6-Aos_4-1	0.96 ± 0.2
	pND6-Aos_4-2	0.85 ± 0.1
	pND6-Aos_5-1	1.09 ± 0.1
	pND6-Aos_5-2	1.23 ± 0.1
	pND6-Aos_7-1	0.96 ± .02
	pND6-Aos_8-2	1.23 ± 0.1
	pND6-Aos_11-5	1.23 ± 0.1

	Note:
	The rubber content is quantified from shoots (leaves + stems) of guayule genotypes grown in MS media.
	Error bars represent three biological plants with three technical replicates each.
	, * and *** indicate significant difference in comparison to G7-11 guayule and/or pND6 (controls) at p > 0.05, 0.005 and 0.0005, respectively.

Next, the genetically altered guayule plants are transplanted into soil and grown for 2 months under 27° C. (16 h)/25° C. (8 h) and 27° C. (16 h)/10° C. (8 h) in growth chamber conditions, representing a microcosm of what guayule plants experience in the field during winter. Under these conditions, pND6-AosiL plants also exhibited elevated rubber content, having up to 31% times more rubber in comparison with G7-11, pND6 and pND6-Aos plants (Table 6). In Table 6, the rubber content is quantified from shoots and roots of the indicated guayule genotypes grown in soil. These plants are approximately 4 months old when rubber content is analyzed (tissue culture (approx. 1.5 months), greenhouse (approx. 1 month), and growth chamber (approx. 2 months)).

TABLE 6

Rubber content of guayule plant tissue determined by Accelerated Solvent Extraction

Average Rubber Content (%)

Shoot

Root

P. argentatum	27° C. (16 h)/	27° C. (16 h)/	27° C. (16 h)/	27° C. (16 h)/
Genotypes	25° C. (8 h)	10° C. (8 h)	25° C. (8 h)	10° C. (8 h)

G7-11.1	1.22 + 0.09	1.13 + 0.11	1.10 + 0.04	0.95 + 0.10
G7-11.2	1.06 + 0.12	1.37 + 0.09	0.63 + 0.12	0.71 + 0.09
pND6-12	1.04 + 0.18	1.31 + 0.06	0.65 + 0.07	0.72 + 0.16
pND6-33	0.90 + 0.06	1.14 + 0.12	0.62 + 0.08	0.82 + 0.10
pND6-35	1.18 + 0.08	1.27 + 0.10	1.09 + 0.06	0.94 + 0.03
pND6-AosiL_7-2	1.49 + 0.07***	1.86 + 0.11***	0.56 + 0.03	1.26 + 0.09**
pND6-AosiL_8-1	1.48 + 0.05***	1.91 + 0.07***	0.78 + 0.14	1.19 + 0.04**
pND6-AosiL_9-16	1.46 + 0.04***	2.01 + 0.08***	0.66 + 0.12	1.16 + 0.02**
pND6-AosiL_12-1	1.55 + 0.07*	1.80 + 0.05**	1.26 + 0.04***	1.52 + 0.07**
pND6-Aos_4-1	0.97 + 0.26	1.13 + 0.2	0.57 + 0.05	0.79 + 0.09
pND6-Aos_4-2	1.21 + 0.10	1.30 + 0.08	1.05 + 0.10	0.94 + 0.11
pND6-Aos_5-1	0.98 + 0.30	1.04 + 0.12	0.54 + 0.11	0.62 + 0.10
pND6-Aos_7-1	0.96 + 0.30	1.15 + 0.14	0.57 + 0.06	0.55 + 0.08

Note:
The rubber content is quantified from shoots and roots of guayule genotypes grown in soil.
Plants are transferred to soil from tissue culture and are grown in a growth chamber environment. Error bars represent three biological plants with three technical replicates each.
, * and *** indicate significant difference in comparison to G7-11 and/or pND6 (controls) at p > 0.05, 0.005 and 0.0005, respectively.

Because rubber is also accumulated in root tissue, the rubber content in the root tissues is also quantified. For the root rubber content, the consistent, significant difference is only under 27° C. (16 h)/10° C. (8 h) in which pND6-AosiL guayule have an increased in rubber content compared with the controls and pND6-Aos (Table 6). The data in Table 6 demonstrate that the combination of cold temperature and silencing PaAos is synergistic, causing guayule to produce a greater amount of rubber than guayule exposed to just cold temperature or to just PaAos silencing. For example, cold treatment alone increased shoot rubber content in the control (pND6-12) by 19%—from an average of 1.04% to 1.24%. But cold treatment of the Aos-downregulated plants (pND6-AosiL) increased rubber by 27%—from average 1.50% to 1.90%. In root tissues, cold treatment increased the rubber content for the control (pND6-12) by 5.1% (from 0.79 to 0.83% rubber), but cold treatment of the Aos-downregulated plants (pND6-AosiL) increased rubber by 57%—from average 0.82% to 1.28%. See Table 7, infra. From the ASE results, the increased in rubber content is very apparent in the pND6-AosiL genotypes.

	TABLE 7

	Average Rubber Content (%)

Shoot

Root

	27° C.	27° C.	27° C.	27° C.
P. argentatum	(16 h)/	(16 h)/	(16 h)/	(16 h)/
Genotypes	25° C. (8 h)	10° C. (8 h)	25° C. (8 h)	10° C. (8 h)

pND6-12	1.04	1.24	0.79	0.83
pND6-AosiL_7-2	1.50	1.90	0.82	1.28
pND6-Aos_4-1	1.03	1.16	0.68	0.73

Example 4. Protein Detection in Rubber Particles

Guayule washed rubber particles (WRPs) are isolated from genetically altered guayule lines (pND6-AosiL and pND6-Aos) and non-altered guayule using the protocol set forth in Cornish and Backhaus, Phytochemistry, 29: 3809-3813 (1990). Rubber particles are extracted from non-altered and genetically altered 1 year old greenhouse plants. First, ˜60 g to ˜70 g of stembark tissues are peeled off from the plant, grounded with a blender containing cold-extraction buffer, and further purified with cold-washed buffer three times by centrifugation. The protein extracts (1 mg) are run on an SDS-PAGE and detected with silver staining. On the SDS-PAGE gel, endogenous Aos protein runs as ˜53 kDa in the non-altered and overexpressed plants but not in the RNAi lines. To determine the dry weight of the WRPs, 50 μL of the protein extracts are aliquoted 3× on a weighing paper, oven-dried over-night in a 60° C. incubator and weighed the next day. Generally, approximately 0.5 mg/μL to approximately 1.5 mg/μL WRPs are extracted.

Example 5. Hormone Production

PaAos is an enzyme in the biosynthetic pathway that produces several different plant hormones, including jasmonic acid, SA, abscisic acid, gibberellin A₂₀, gibberellin A₁, and gibberellin A₃. As such, the amount of these hormones is quantified in genetically altered (pND6-AosiL and pND6-Aos), empty vector transformed (pND6 without PaAos; control), and wild-type (G7-11, control) tissue-cultured guayule plants using the protocol described in Pan et al., Nature Protocols 5:986-992 (2010). See Table 8, infra. Briefly, leaves and stems are snap-frozen and ground to powder with mortar and pestle. Solvent extraction solution containing 2-propanol/H₂O/concentrated HCl (2:1:0.002; vol/vol/vol) and internal standards are added to ˜50 mg of pre-weighed tissues. After solvent extraction, sample concentration and re-dissolution, 50 μL of the sample solution is placed into the liquid chromatography-tandem spectrometry (Agilent GC-MS 5977A; Agilent Technologies, Santa Clara, Calif.) for hormone analysis. Three biological plants, with three technical replicates of each plant, are used.

	TABLE 8

	Concentration (ng/gfw)

P. argentatum	Jasmonic	Salicylic	Abscisic	Gibberellin	Gibberellin	Gibberellin
Genotypes	Acid	Acid	Acid	A₂₀	A₁	A₃

G7-11	5.36 ± 1.2	5.50 ± 0.8	11.01 ± 1.9	15.95 ± 0.7	9.95 ± 0.07	3.52 ± 0.2
pND6-12	1.57 ± 0.1	4.89 ± 0.6	7.05 ± 0.8	14.11 ± 1.2	12.39 ± 2.2	2.19 ± 0.01
pND6-33	4.76 ± 1.0	5.04 ± 0.1	7.24 ± 0.3	13.86 ± 2.6	12.70 ± 2.3	1.79 ± 0.09
pND6-35	1.96 ± 0.4	5.6 ± 0.5	7.10 ± 0.6	14.41 ± 1.2	14.65 ± 0.2	2.16 ± 0.3
pND6-AosiL_7-2	0.57 ± 0.1**	9.51 ± 0.5*	4.71 ± 0.6*	9.63 ± 1.2*	5.13 ± 0.8*	0.86 ± 0.3*
pND6-AosiL_9-16	0.57 ± 0.01**	7.65 ± 0.2*	2.96 ± 0.3*	8.85 ± 2.1*	7.81 ± 0.2**	0.80 ± 0.3*
pND6-AosiL_12-1	0.68 ± 0.05**	9.65 ± 1.1*	3.64 ± 0.5*	10.59 ± 0.2*	6.75 ± 1.1*	1.32 ± 0.07*
pND6-Aos_4-1	1.48 ± 0.2	4.03 ± 0.7	9.13 ± 1.71	15.49 ± 0.6	10.78 ± 0.1	1.93 ± 0.06
pND6-Aos_4-2	3.25 ± 0.2	4.76 ± 0.6	13.9 ± 1.3	no data	14.63 ± 2.7	1.80 ± 0.1
pND6-Aos_7-1	1.41 ± 0.3	5.50 ± 0.02	8.84 ± 0.3	13.64 ± 1.2	13.96 ± 0.01	1.84 ± 0.1

± represent three biological plants with three technical replicates each plant.
* and ** indicate significant difference in comparison to G7-11 and/or pND6 (controls) at p > 0.05 and 0.005, respectively.

As evident in Table 8, the amount of jasmonic acid, abscisic acid and gibberellic acids are reduced in the pND6-AosiL guayule compared to the amount in the controls (wild-type (G7-11) and empty vector transformed plants) and pND6-Aos guayule. Conversely, the SA content is elevated in pND6-AosiL compared to the controls and pND6-Aos lines. These results suggest that knocking down PaAos expression not only reduces production of jasmonic acid but also affects the level of other hormones as well.

Example 6. Plant Architecture and Photosynthetic Rates

Three independent events from each of the overexpression (pND6-Aos) and of the silenced (pND6-AosiL) lines; as well as two pND6 and one wild-type (G7-11) controls are selected for further studies. pND6-AosiL plants grown in greenhouse (data not shown) and growth chamber conditions are bigger (see FIG. 3), have darker green leaves (data not shown), and increased chlorophyll measurement than the wild type and other genetically altered plants (see FIG. 4). As demonstrated in FIG. 3, under 27° C. (16 h)/25° C. (8 h) and 27° C. (16 h)/10° C. (8 h) environments, pND6-AosiL plants are significantly taller and wider in both conditions. These plant architectural traits reflect the fact that pND6-AosiL plants are larger and have more shoot and root biomass.
pND6-AosiL genotypes have also a greater number of stems than the wild-type and empty vector controls. Well-branched guayule plants are an indicator of having increased rubber yield because of the presence of more sink tissue available to store rubber. Additionally, the mature stembark tissues in pND6-AosiL have thicker diameter (ranging from approximately 35% to approximately 54%) under both 27° C. (16 h)/25° C. (8 h) and 27° C. (16 h)/10° C. (8 h) in comparison to the controls and pND6-Aos. See FIG. 5.
Based on this observation, the photosynthetic rate of the plants is measured using LI-COR 6400xt (LI-COR Biosciences, Lincoln, Nebr.) to measure the photosynthetic rate. Measurements are taken between 0900 to 1200 h. Fully expanded middle leaf are clamped on the Li-Cor head. After the measured and set parameters are stabilized, the reading is taken. The middle leaf position is chosen because this position shows significant differences based on chlorophyll meter measurements, FIG. 4. (SPAD-502; Minolta Camera Ltd., Japan). The pND6-AosiL plants exhibit higher photosynthetic rate (23-31%) in comparison to G7-11, pND6 and pND6-Aos plants (Table 9, infra). Additional physiological measurements reveal that pND6-AosiL stomatal limitation is one of the factors involved in the higher photosynthetic rate as pND6-AosiL plants show higher stomatal conductance and transpiration rate when compared to G7-11, pND6 and pND6-Aos plants (Table 9, infra). Furthermore, chlorophyll fluorescence measurements clearly show PSII and ETR parameters used for elucidating the efficiency of PSII are significantly higher than G7-11, pND6 and pND6-Aos plants (Table 9, infra) in both 27° C. (16 h)/25° C. (8 h) and 27° C. (16 h)/10° C. (8 h) treatment conditions. Having higher PSII and ETR indicate that amount of light energy absorbed and carbon assimilated is available more in pND6-AosiL plants to convert into energy for the plant to use (i.e., growth and development as well as rubber production) compared to the controls and pND6-Aos plants. Meanwhile, the NPQ measurements for the pND6-AosiL plants are lower under the 27° C. (16 h)/25° C. (8 h) condition and higher under the 27° C. (16 h)/10° C. (8 h) treatment in comparison with the controls and pND6-Aos plants. Having higher NPQ suggests that pND6-AosiL have improved heat dissipation ability compared to G7-11, pND6 and pND6-Aos plants which could help prevent lipid or other cell membrane damage under environmental stress.

27° C. (16 h)/25° C. (8 h)

G7-11	5.75 ± 0.8	0.093 ± 0.03	2.33 ± 0.6	0.146 ± 0.015	115.73 ± 11.3	1.97 ± 0.2
pND6-10	6.29 ± 0.7	0.110 ± 0.03	2.74 ± 0.6	0.141 ± 0.015	111.1 ± 11.6	1.74 ± 0.1
pND6-12	6.20 ± 0.8	0.109 ± 0.02	2.66 ± 0.5	0.145 ± 0.015	114.4 ± 12.1	1.90 ± 0.2
pND6-AosiL_7-1	8.56 ± 0.6***	0.147 ± 0.01***	3.41 ± 0.3***	0.196 ± 0.027*	165.2 ± 10.7***	1.33 ± 0.2***
pND6-AosiL_8-1	8.40 ± 0.6***	0.155 ± 0.02***	3.67 ± 0.4***	0.180 ± 0.008*	141.4 ± 6.2**	1.27 ± 0.2***
pND6-AosiL_9-16	7.96 ± 0.5***	0.162 ± 0.03***	3.57 ± 0.5***	0.186 ± 0.018*	134.4 ± 3.3**	1.19 ± 0.3***
pND6-Aos_4-1	5.62 ± 0.9	0.110 ± 0.04	2.59 ± 0.7	0.133 ± 0.008	104.7 ± 6.7	1.92 ± 0.3
pND6-Aos_5-1	5.70 ± 0.8	0.110 ± 0.04	2.60 ± 0.7	0.133 ± 0.008	104.7 ± 6.7	1.95 ± 0.3
pND6-Aos_7-1	5.87 ± 0.9	0.113 ± 0.04	2.65 ± 0.7	0.137 ± 0.008	107.7 ± 6.3	2.07 ± 0.4

27° C. (16 h)/10° C. (8 h)

G7-11	2.23 ± 0.5	0.054 ± 0.02	1.31 ± 0.4	0.070 ± 0.007	55.4 ± 5.4	1.58 ± 0.2
pND6-10	2.04 ± 0.4	0.057 ± 0.02	1.55 ± 0.3	0.064 ± 0.006	50.7 ± 4.9	1.56 ± 0.2
pND6-12	2.28 ± 0.4	0.065 ± 0.03	1.67 ± 0.7	0.066 ± 0.003	51.6 ± 2.7	1.51 ± 0.3
pND6-AosiL_7-1	4.14 ± 0.4***	0.104 ± 0.02***	2.54 ± 0.4***	0.104 ± 0.011***	81.0 ± 8.2***	2.29 ± 0.3**
pND6-AosiL_8-1	4.15 ± 0.4***	0.112 ± 0.05***	2.71 ± 0.8***	0.102 ± 0.013***	77.0 ± 4.5***	2.33 ± 0.2**
pND6-AosiL_9-16	4.14 ± 0.6***	0.101 ± 0.03***	2.60 ± 0.5***	0.101 ± 0.009***	79.4 ± 6.9***	1.95 ± 0.1**
pND6-Aos_4-1	2.27 ± 0.5	0.059 ± 0.02	1.51 ± 0.3	0.065 ± 0.011	60.4 ± 5.5	1.35 ± 0.3
pND6-Aos_5-1	2.97 ± 0.3	0.069 ± 0.01	1.81 ± 0.2	0.077 ± 0.007	58.5 ± 6.3	1.25 ± 0.2
pND6-Aos_7-1	2.45 ± 0.6	0.063 ± 0.01	1.93 ± 0.3	0.065 ± 0.010	50.1 ± 5.8	1.44 ± 0.2

Pn = net photosynthetic rate;
g = stomatal conductance;
€ = Transpiration rate;
ΦPSII = Efficiency of Photosystem II;
ETR = Electron Transport Rate;
NPQ = Non-photochemical quenching
, * and ***indicate significant difference in comparison to G7-11 and/or pND6 (controls) at p > 0.05, 0.005 and 0.0005, respectively.

Example 7. Quality of Natural Rubber from pND6-AOSiL Plants

The length of the polymer chain, a.k.a. rubber molecular weight, is the primary determinant of quality in natural rubber. Gel permeation chromatography (GPC) is used to measure the molecular weight of rubber from guayule tissue culture plants' extracts. Cyclohexane extractables collected from ASE (see Example 3 and Table 5 supra) are re-suspended in approximately 3 mL of tetrahydrofuran (THF) overnight with gentle shaking (Multi-Purpose Rotator. Thermo Scientific, Waltham. Mass.). The solution is syringe-filtered through a 1.6 μm glass microfiber GF/A filter (Whatman GE Healthcare, Piscataway, N.J.), then injected into a Hewlett Packard 1100 series HPLC (1.0 mL/min flow rate, 50 μL injection volume, THF continuous phase) and size exclusion separated by two Agilent PL gel 10 μm Mixed-B columns in series (35° C.) (Santa Clara, Calif.). The resulting chromatograms are used to calculate the rubber molecular weight shown in FIG. 6 (using Astra software (Wyatt Technology Corp., Santa Barbara, Calif.)). The molecular weight of natural rubber from three pND6-AosiL transformed guayule plants (silenced) is greater than from wild-type guayule line G7-11, two negative control pND6 transformed guayule plants and three pND6-Aos transformed guayule plants (overexpressed) indicating better quality rubber in the PaAos silenced guayule plants. In FIG. 6, the asterisks (*) and (**) above the three pND6-AosiL transformed guayule plant lines indicate significant difference in comparison to the negative control pND6 transformed guayule plant lines at p>0.05 and p>0.005, respectively.

Example 8. PaAos SNPs Change Protein's Functionality

Eight different guayule cultivars grown in tissue culture are evaluated for their rubber content and expression of PaAos gene. The combination of ASE method (see Example 3 supra) and qRT-PCR (see the protocols and primers discussed in Example 2, supra, and FIG. 2) are used to compare rubber content to the level of PaAos gene expression. First, seeds from guayule lines (PI 478648, W6 549, PI 478651, PI 478652, PI 478653, PI 478654, PI 478655, and PI 478662) are obtained from a public germplasm bank (USDA-ARS National Plant Germplasm System, Parlier, Calif.). The seeds are germinated and plants grown in tissue culture medium for 8 weeks. The natural rubber content is determined by standard methods, as described previously (ASE). The rubber content varied significantly between lines, from 0.95% to 1.73% (Table 10). Cultivar (line) W6 549 has the lowest average rubber content (%) and cultivar PI 478652 has the largest average rubber content (%) (see Table 10).

TABLE 10

P. argentatum	Germplasm
Genotypes	name	Average Rubber Content (%)

PI 478648	11635	1.38 ± 0.08
W6 549	CAL 7	0.95 ± 0.03
PI 478651	11701	1.40 ± 0.13
PI 478652	12229	1.73 ± 0.28
PI 478653	12231	1.36 ± 0.11
PI 478654	N396	1.20 ± 0.05
PI 478655	N565	0.98 ± 0.01
PI 478662	A48118	1.07 ± 0.06

PaAos gene expression for cultivars W6 549 and PI 478652 are determined by standard methods (qRT-PCR, see Example 2, supra). Shoot tissues (leaf and stem) are collected into 2 mL tubes and are snap-frozen in liquid nitrogen and then hand pulverized (mortar and pestle). RNA is extracted using the TRIzol® method (Ambion, Pittsburgh, Pa.) using manufacturer's recommended protocol. The RNA concentration is quantified with the NanoDrop ND1000 (ThermoScientific, Wilmington, Del.). An RNA cleanup is performed using RNeasy MinElute Cleanup kit using manufacturer's recommended protocol (Qiagen Inc., Valencia, Calif.). The RNA is eluted with 30-50 tit of RNase-free water along with on-column DNase1 treatment.
An iScript cDNA synthesis kit (Bio-Rad, Hercules, Calif.) is used to synthesize complementary DNA (cDNA) from the isolated RNA. An amount of 1 μg of RNA is used in the 20 mL reaction mixture. For semi-qRT-PCR, 2 μL of the diluted cDNA (1:20) is used in a 50 μL reaction mixture of One Taq Quick-Load 2× Master Mix with Standard Buffer (New England Biolabs, Inc., Ipswich, Mass.) with the following forward and reverse primers (10 μM) 5′-ATGGACCCATCGTCTAAACCC-3′ (SEQ ID NO: 16) and 5′-TCATATACTAGCTCTCTTCAGG-3′ (SEQ ID NO: 17), respectively. The semi-qRT-PCR is run using Eppendorf thermocyler (ThermoFisher Scientific, Waltham, Mass.) with the following thermal cycle: 94° C. pre-incubation for 30 seconds; amplification for 35 cycles at 94° C. for 30 seconds, annealing at 58° C. for 1.5 minutes, and extension at 68° C. for 1 minute. The final extension time is for 5 minutes at 68° C. Each semi-qRT-PCR run is performed with three independent tissue samples. The 18S gene (˜200 bp) is used as an internal control using SEQ ID NO: 5 as forward primer and SEQ ID NO: 6 as reverse primer. PCR products (˜1.4 Kbp) are separated by electrophoresis on a 1% (w/v) agarose gel. Results clearly demonstrate that W6 549 (“W6549”) cultivar has increased PaAos expression compared to PI 478652 (“478652”) cultivar (see FIG. 7). These results suggest that the rubber content is inversely correlated with the PaAos gene expression.
PaAos coding sequence in the two lines, W6 549 cultivar (low rubber producer) and PI 478652 cultivar (high rubber producer) are determined by PCR, and the sequences are compared to G7-11 cultivar sequence. Extraction of the cDNA from the agarose gel is performed with QIAquick Gel Extraction kit (Qiagen, Germantown, Md.) using manufacturer's recommended protocol. The 1.4 kb band visualized with ethidium bromide is excised from the gel with a clean razor blade. After determining the weight of the gel slice, 300 μl of Buffer QG pH 7.5 is added for every 100 mg of gel slice with the DNA fragment size of 100 bp-4 kb. To bind the DNA, 30 μl QIAEX II beads are added per 5 μg of DNA. The resuspended gel is dissolved by incubation at 50° C. for 10 minutes with vigorous vortexing every 2 minutes. Each sample is centrifuged at 16,110×g in a conventional table top microcentrifuge for 30 seconds. After centrifugation, each sample rests at room temperature for five minutes. The supernatant is discarded, and the pellet is washed with cold 750 μl Buffer PE twice. The pellet is air dried until it turned solid white. The DNA is resuspended with 50 μl of 10 mM of Tris-Cl pH 8.5. The dissolved DNA pellet stands at room temperature for 1 minute prior to centrifugation. The supernatant is collected as the purified cDNA product. To confirm the integrity of the sequence, three independent PCR products are sent to Elim Biopharmaceuticals (Hayward, Calif.) for analysis. The sequence alignment is performed using softwares MEGA 6.06 (Tamura, et al., Mol. Bio. and Evol., 30:2725-2729 (2013)) and T-Coffee (Notredame, et al., J. Mol. Biol., 302:205-217 (2000)). As evident in FIGS. 8A-8C, a few SNPs exist which give rise to changes in the amino acid sequences (see FIG. 9). In particular, the amino acids at positions 318, 408 and 459 are D, I and L in W6 549 (“W6549”) cultivar while PI 478652 (“478652”) cultivar has N, V and W, respectively (FIG. 9). These differences in three amino acids result in different PaAos functionality which result in different amounts of rubber being produced.
By reducing PaAos' functionality, one increases rubber production in guayule. As discussed previously, reducing the amount of PaAos by silencing PaAos expression or translation increases rubber production. Null mutations (such as, but not limited to, insertions that disrupt translation of a functional protein, changing slice site recognition nucleotide(s), and changing ATG initiation codon) alter the production of functional PaAos which result in an increase in rubber production. Alternations in PaAos' DNA sequence that result in specific amino acid changes within PaAos also increase rubber production. For example, DNA alterations that change PaAos sequence from D318, I408 and/or L459 (present in W6 549 cultivar, low rubber producer) to N318, V408 and W459 (present in PI 478652 cultivar, high rubber producer) (or any other non-conservative amino acid for D318, I408, and/or L459) result in an increase in rubber production because of a decrease in PaAos functionality. In addition, altering PaAos' DNA sequence encoding S332, E336, R339, S359, and/or S411 to a sequence encoding non-conservative amino acids results in reducing PaAos' functionality and thus increasing rubber production. See, Pan, et al., J. of Bio. Chem., 273(29):18139-18145 (1998), contents of which are expressly incorporated by reference.
Based on these results from these assays, one can screen for the presence of particular SNPs in the PaAos gene in various guayule varieties at the seedling stage to determine if a particular guayule variety is a high rubber producer or low rubber producer. To screen guayule, one obtains a tissue sample from the guayule to be screened, isolates the sample's mRNA or total RNA, and conduct a PCR assay (regular PCR or RT-PCR or qRT-PCR) using PaAos primers that surround the nucleotides encoding amino acids N318, V408 and W459 to identify guayule plants encoding these amino acids which indicate that the guayule produces more rubber than a guayule not having these amino acids within PaAos. Guayule seedlings (plants that are between 2-4 weeks and 8-10 weeks post-germination) are screened. Alternatively, guayule plants that are approximately 2 or 3 months old can be screened. While any plant tissue can be used to conduct the SNP analysis, bark and leaves may be easier to sample than other tissue (such as roots). Using the two primer pairs 5′-CCTACTCGACGCCAAGAG-3′ (forward, SEQ ID NO: 18) and 5′-TTCAGCTGAGCATGTCTAGGT-3′ (reverse, SEQ ID NO: 19) and 5′-GGCATTGTTGAAGTACATATGG-3′ (forward, SEQ ID NO: 20) and 5′-CCAAAGGAGACTCGCCTAATT-3′ (SEQ ID NO: 21), one determines if the seedling or plant contains D318, I408 and/or L459 (similar to G7-11 and W6 549 cultivars) and thus produces less rubber than a “high rubber producer” plant. Alternatively, using the two primer pairs 5′-CCTACTCGACGCCAAAAGC-3′ (forward, SEQ ID NO: 22) and 5′-CTTAAGTTGAGCATGTCTAGGTT-3′ (reverse, SEQ ID NO: 23) and 5′-GGCATTGTTGAAGTACGTATGG-3′ (forward, SEQ ID NO: 24) and 5′-CCCAAGGAGACTCGCCTA-3′ (reverse, SEQ ID NO: 25), one determines if the seedling or plant contains N318, V408 and/or W459 (similar to PI 478652 cultivar) and thus produces more rubber than a “low rubber producer” plant. Furthermore, guayule containing PaAos with S332, E336, R339, S359, and/or S411, in combination with one or more of D318, I408 and L459, are also low rubber producing plants. Primers are designed to cover the SNPs for these amino acids which are used to identify low rubber producing guayule. Similarly, guayule containing PaAos with non-conservative amino acids to D318, S332, E336, R339, S359, I408, S411, or L459, or a combination thereof, are high rubber producing plants, and primers are designed to cover the SNPs for these amino acids which are used to identify high rubber producing plants. After performing the PCR assay, one isolates the amplicon(s) and sequences the amplicon(s) to determine the presence or absence of the indicated SNPs. Other techniques are known to one of ordinary skill in the art for identifying amplicons with the indicated SNPs.
Alternatively, an ELISA or other type of antibody assay can distinguish between PaAos containing N318, V408 and/or W459 (PI 478652 cultivar (high producer)) and PaAos containing D318, I408 and/or L459 (W6 549 cultivar (low producer)), with or without one or more of S332, E336, R339, S359, and S411. An ELISA using a monoclonal antibody (mAb) that is specific for PaAos containing N318, V408 and/or W459, with or without one or more non-conservative amino acids substituted for S332, E336, R339, S359, and S411, would identify high rubber producing plants. Alternatively, an ELISA using a mAb that is specific for PaAos containing D318, I408 and/or L459 with or without one or more amino acids S332, E336, R339, S359, and S411, would identify low rubber producing plants. Protein isolated from tissue sample, as described above, can be contacted with the mAb(s) in the ELISA which then changes color to indicate the presence of PaAos having the particular amino acids and structure to which the mAb binds.
Guayule encoding PaAos with conservative amino acid substitutions for N318, V408 and/or W459 (and optionally with non-conservative amino acid substitutions for S332, E336, R339, S359, and/or S411) are high rubber producing guayule. Similarly, guayule encoding PaAos with conservative amino acid substitutions for D318, I408 and/or L459 (and optionally with or without conservative amino acid substitutions for S332, E336, R339, S359, and/or S411) are low rubber producing guayule. See Table 2 and preceding paragraph for information about conservative and non-conservative amino acid substitutions, and Table 1 for DNA codons for amino acids.
One can generate DNA mutations within guayule seed's genome using EMS, UV light, protons, or other known mutagens to create altered guayule seeds. Then one germinates the seeds into seedlings and screen the seedlings for PaAos mutations which reduce PaAos' functionality. In one embodiment, the above described primers are used to determine if the indicated SNPs are present in the altered guayule seedling so that one does not need to grow the altered guayule for years before determining if the altered guayule is likely a high rubber producer or a low rubber producer. One can screen for D318, S332, E336, R339, S359, I408, S411, and/or L459 and conservative amino acids to determine if the altered seedling is a low rubber producer; or screen for non-conservative amino acid substitutions to determine if the altered seedling is a high rubber producer.
The foregoing detailed description and certain representative embodiments and details of the invention have been presented for purposes of illustration and description of the invention. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. It will be apparent to practitioners skilled in the art that modifications and variations may be made therein without departing from the scope of the invention. All references cited herein are incorporated by reference.

P. argentatum

We, the inventors, claim as follows:

1. An altered guayule, parts and progeny thereof, that produces more rubber than amount of rubber produced by a non-altered guayule comprising a mutation in PaAos; wherein said mutation is selected from the group consisting of (i) an alteration in a PaAos codon encoding an amino acid to a stop codon, (ii) an alteration of PaAos' translation initiation codon to another codon, (iii) an alteration in PaAos ribosome binding site's sequence, (iv) an alteration in one or more PaAos splice site codons, (v) a deletion of part or all of said PaAos' sequence, (vi) an insertion of DNA into PaAos, and (vii) an alteration in one or more PaAos DNA codon sequences to encode a non-conservative amino acid; wherein said mutation reduces said altered PaAos' functionality compared to amount of PaAos functionality in said non-altered guayule; wherein said reduced PaAos functionality causes said altered guayule to produce an increased amount of rubber compared to said amount of rubber produced by said non-altered guayule.

2. The altered guayule of claim 1; wherein said alteration in one or more PaAos DNA codon sequences to encode a non-conservative amino acid occurs at amino acids located at 318, 332, 336, 339, 359, 408, 411, and 459 with said PaAos sequence.

3. The altered guayule of claim 2; wherein said non-conservative amino acid substitution excludes at least one of N318, V408 and W459.

4. An altered cell of said altered guayule of claim 1, wherein said altered cell comprises said mutation in PaAos.

5. An altered germplasm of said altered guayule of claim 1, wherein said altered germplasm comprises said mutation in PaAos.

6. An altered seed of said altered guayule of claim 1, wherein said altered seed comprises said mutation in PaAos.

7. A method for producing an altered guayule that comprises a mutated PaAos and produces an increased amount of rubber compared to amount of rubber produced by a non-altered guayule, said method comprising

exposing a non-altered guayule cell or seed to a mutagen to produce a mutated guayule cell or seed with said mutated PaAos;

selecting said mutated guayule cell or seed comprising said mutated PaAos, wherein said mutated PaAos encodes an altered PaAos having reduced functionality compared to a non-altered PaAos' functionality; and

growing said selected mutated guayule cell or seed comprising said mutated PaAos to produce an altered guayule that produces said altered PaAos with reduced functionality and said increased amount of rubber compared to said amount of rubber produced by said non-altered guayule.

8. The method of claim 7, wherein said mutated PaAos comprises at least one of (i) an alteration in a PaAos codon encoding an amino acid to a stop codon, (ii) an alteration of PaAos' translation initiation codon to another codon, (iii) an alteration in PaAos ribosome binding site's sequence, (iv) an alteration in one or more PaAos splice site codons, (v) a deletion of part or all of said PaAos' sequence, (vi) an insertion of DNA into PaAos, and (vii) an alteration in one or more PaAos DNA codon sequences to encode a non-conservative amino acid.

9. The method of claim 8, wherein said alteration in one or more PaAos DNA codon sequences to encode a non-conservative amino acid occurs at amino acids located at 318, 332, 336, 339, 359, 408, 411, and 459 with said PaAos sequence.

10. An altered cell of said altered guayule produced according to said method of claim 7.

11. An altered germplasm of said altered guayule produced according to said method of claim 7.

12. An altered seed of said altered guayule produced according to said method of claim 7, wherein said altered seed comprises said mutated PaAos.

13. A method of producing a population of high rubber producing guayule plants or seeds comprising PaAos with low functionality, said method comprising:

genotyping a first population of guayule plants or seeds, said first population of said guayule plants or seeds comprising said PaAos with low functionality;

selecting from said first population one or more guayule plants or seeds comprising said PaAos with low functionality based said genotyping; and

producing from said selected one or more guayule plants or seeds comprising said PaAos with low functionality a second population of guayule plants or seeds comprising said PaAos with low functionality.

14. The method of claim 13, wherein said PaAos with low functionality comprises at least one amino acid selected from group of N318, V408, W459, conservative amino acids substitutions thereof, and non-conservative amino acid substitutions at S332, E336, R339, S359, and S411.

15. A method of identifying a high rubber producing guayule comprising detecting PaAos with low functionality in a test guayule, wherein when said test guayule contains PaAos with amino acids N318, V408 and W459, then said test guayule is a high rubber producing guayule.

16. The method of claim 15, wherein said detecting step comprises

contacting said PaAos from said test guayule with monoclonal antibodies that bind to PaAos with amino acids N318, V408 and W459; and

determining if said monoclonal antibodies binds to said test guayule PaAos.

17. The method of claim 15, wherein said detecting step comprises

obtaining nucleic acids from said test guayule;

performing a PCR assay with said obtained nucleic acids, primer sets having SEQ ID NOs: 22 and 23 and SEQ ID NOs: 24 and 25, and a label;

determining if an amplicon is generated, wherein when said amplicon is produced, then said test guayule contains a PaAos that encodes a PaAos having amino acids N318, V408 and W459, and said test guayule is a high rubber producing guayule.