EP1442053A2

EP1442053A2 - New protein of the defensin family from oil palm

Info

Publication number: EP1442053A2
Application number: EP02785408A
Authority: EP
Inventors: Suan Choo Persiaran Institusi CHEAH; James W. Cirad CP/IRD Oil Palm Biotech. TREGEAR
Original assignee: Centre de Cooperation Internationalel en Recherche Agronomique pour le Development CIRAD; Malaysian Palm Oil Board MPOB
Current assignee: Centre de Cooperation Internationalel en Recherche Agronomique pour le Development CIRAD; Malaysian Palm Oil Board MPOB
Priority date: 2001-11-06
Filing date: 2002-11-05
Publication date: 2004-08-04
Also published as: WO2003040297A2; AU2002350712A1; WO2003040297A3; TW200407423A

Abstract

The invention relates to new protein of the defensin family from oil palm, its isolated polypeptide and an isolated polynucleotide encoding it. The invention further relates to the uses of the polynucleotide and or polypeptide, including its use to detect the mantled abnormality of oil palm.

Description

NEW PROTEIN OF THE DEFENSIN FAMILY

The present invention relates to a new protein of the defensin family, which is useful in particular as a molecular marker of somaclonal variation events associated with the mantled homeotic flowering abnormality of oil palm.

Oil palm (Elaeis guineensis Jacq.) is a diploid onocotyledonous temporally dioecious species producing alternate cycles of male and female flowers.

In vi tro micropropagation based on somatic embryogenesis has been used to carry out the multiplication of elite oil palm genotypes as described by RIVAL et al . , Plan Tiss. Cult. Biotechnol., 3, 74-83, 1996; however, the large scale use of this approach is hampered by the appearance of a somaclonal variant, known as mantled as described by CORLEY et al . , The Planter, 62, 233-240, 1986, which affects approximately 5-10% of palms cloned in this way as illustrated again by RIVAL et al . , Plan Tiss. Cult. Biotechnol., 3, 74-83, 1996). The mantled phenotype involves a feminisation of both male and female flowers: in the former, stamens develop as carpelloid structures, whilst in the latter, the staminodes (vestigial stamens) develop as pseudocarpel structures. In both cases, the petals appear to develop as sepalloid structures. In severe cases the flowers are sterile, although lesser affected female -flowers may be fertilised to give mantled fruit.

In order to allow a more widespread use of in vi tro micropropagation for oil palm, it is necessary to improve its reliability, by establishing an early detection method for the mantled abnormality. To this end, a detailed study has been carried out in order to identify molecular markers of the mantled phenotype.

Previous work revealed that the mantled abnormality is epigenetic in nature. Firstly, it was observed that reversion to a normal floral phenotype may occur in the field as described by DURAND-GASSELIN T. et al., Oleagineux 45, 1-11, 1990. Secondly, although the mantled abnormality is strongly transmitted through tissue culture, only a weak non- Mendelian transmission occurs via seeds as described by RAO V. and DONOUGH C.R., Elaeis, 2 199-207, 1990. Thirdly, ploidy, RAPD and AFLP studies have shown that the abnormal phenotype is unlikely to have resulted from any gross changes in genomic structure as described by RIVAL et al . , Plant Cell. Rep., 16, 884-887, 1997; RIVAL et al., Plant Breeding, 117, 73-76, 1998.

The present invention is based on the cloning and characterisation of a new oil palm gene, hereinafter referred as EGAD1 . The EGAD1 gene codes for a 77 amino acid polypeptide displaying strong similarities with plant defensins.

Northern studies of EGAD1 gene expression show that in the intact oil palm plant, EGAD1 transcripts accumulate mostly in inflorescence tissues, both normal and man tled, and show a peak of abundance during the early stages of development of the male inflorescence. On the other hand, in tissue culture, EGADl transcripts are observed to accumulate throughout the regeneration procedure used for oil palm, a higher level of expression being observed at the nodular callus stage in cultures carrying the mantled abnormality than in those lacking it.

Thus, the expression of EGADl provides an early marker of the mantled phenotype.

The present invention relates to an isolated polypeptide of the defensin family, wherein said polypeptide, hereinafter referred as EGADl, has at least 70% sequence identity, preferably at least 80% sequence identity, and more preferably at least 90% sequence identity, with the polypeptide of SEQ ID NO: 2 in the enclosed sequence listing. The percent sequence identity is based on the entire region SEQ ID NO: 2 and is determined according to the CLUSTAL W program as illustrated by THOMPSON et al . , Nucl. Acids. Res., 22, 4673-4680, 1994, using the default settings.

The invention also comprises fragments of an EGADl polypeptide of the invention, comprising at least 5, preferably 10 contiguous amino-acids of said polypeptide.

The term "polypeptide" herein refers to a polymer of amino acid residues, resulting from the translation of a nucleic acid sequence, and possibly having post-translational modifications such as glycosylation, lipid attachment, hydroxylation, etc...

The present invention also relates to an isolated polynucleotide selected among: a) a polynucleotide encoding an EGADl polypeptide; this includes for instance EGADl genes, EGADl cDNA, or EGADl mRNA. A particular example is the cDNA of SEQ ID NO: 1; b) a fragment of a polynucleotide a) at least 10, preferably at least 15, and more preferably at least 20 contiguous nucleotides thereof ; c) a polynucleotide complementary to a polynucleotide a) or b) above.

The invention also comprises polynucleotides hybridising selectively under stringent conditions with any polynucleotide of the invention. This includes in particular nucleic acid probes for detecting a nucleic acid sequence encoding an EGADl polypeptide, as well as nucleic acid primers for amplifying a nucleic acid sequence encoding an EGADl polypeptide, or a portion thereof.

The term "stringent conditions" refers to conditions under which a polynucleotide will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold, preferably at least 5-fold over background) . Stringent conditions can easily be defined by one of skill in the art for a polynucleotide of given length and base composition.

According to a preferred embodiment, a nucleic acid probe of the invention consists essentially of a polynucleotide of at least 10, preferably at least 15, and more preferably at least 20 nucleotides hybridising selectively under stringent conditions with a nucleic acid of SEQ ID NO: 1. Said probe may further comprise modifications, such as the addition of an appropriate label allowing its detection.

According to a preferred embodiment, a set of PCR primers of the invention consists of a fragment of at least 10, preferably at least 15, and more preferably at least 20 contiguous nucleotides of the polynucleotide of SEQ ID NO: 1, and a fragment of at least 10, preferably at least 15, and more preferably at least 20 contiguous nucleotides of the complementary of SEQ ID NO: 1. Appropriate primers can easily be designed by one of skill in the art from the sequence SEQ ID NO: 1.

The invention also encompasses any polynucleotide obtained from an oil palm genomic or cDNA library by PCR amplification with a set of primers of the invention.

The invention also comprises: - a recombinant expression cassette comprising a polynucleotide of the invention encoding an EGADl polypeptide operably linked to a promoter;

- a recombinant nucleic acid vector having an insert consisting of a polynucleotide or of a recombinant expression cassette of the invention;

- a host cell transfected with a polynucleotide or a recombinant expression cassette of the invention; a transgenic plant comprising a recombinant expression cassette of the present invention. The invention also provides a process for producing an EGADl polypeptide of the invention, wherein said process comprises culturing a host cell transfected with a recombinant expression cassette of the invention, and recovering the EGADl polypeptide from said culture. Said polypeptide can be obtained from the cell lysate or from the culture medium using standard protein isolation techniques, in particular techniques suitable for isolation of defensins.

The invention also comprises polyclonal or monoclonal antibodies specifically directed against an EGADl polypeptide of the invention.

The present invention also relates to the use of a polypeptide or a polynucleotide of the invention, or of antibodies of the invention, for detecting the man tled phenotype in oil palm tissue, or in an oil palm tissue culture.

More specifically, the present invention provides a method for detecting the man tled abnormality in an oil palm tissue culture, wherein said method comprises detecting the overexpression of the EGADl gene encoding the EGADl polypeptide in said culture.

According to a preferred embodiment of the invention, the overexpression of the EGADl gene is detected in an oil palm tissue culture at the callus stage.

The overexpression of the EGADl gene is herein defined as a level of expression of said gene higher than the base-line expression level in a normal oil palm tissue culture . Generally, the level of expression of the EGADl gene in oil palm tissue culture bearing the mantled abnormality is at least 2 times higher, preferably at least 3 times higher, and more preferably at least 4 times higher, than its level of expression in a normal oil palm tissue culture.

The level of expression of the EGADl gene can be determined by evaluating the amount of EGADl transcripts, or the amount of EGADl polypeptide.

Techniques allowing the detection of an increased production of a mRNA or a protein are well known in the art.

An increased production of mRNA may for instance be detected using the technique of northern blot analysis. An increased production of EGADl polypeptide may for instance be detected by immunoassay using antibodies of the invention directed against said polypeptide.

The invention also encompasses reagents for practising the method of the invention. These reagents include in particular nucleic acid probes and sets of PCR primers as defined above. The EGADl gene and the EGADl polypeptide can also be used in the same way as previously described for other plant defensins (THEVISSEN et al . , J. Biol. Chem. 271, 15018- 15025, 1996; GAO et al. Nature Biotech., 18, 1307-1310 (2000); US Patent 6,121,436; (TERRAS et al . , Plant Cell, 7, 573-588, 1995)), for the purpose of protecting plants against pathogens, in particular microorganisms such as fungi.

The use of the EGADl gene and the EGADl polypeptide to protect plants against pathogens can be achieved by standard methods, for instance by transforming the plant to be protected with a sequence encoding an EGADl polypeptide, placed under the transcriptional control of an appropriate promoter.

The present invention will be further illustrated by the additional description which follows, which refers to the isolation of the EGADl cDNA and the use of EGADl sequences for detecting the mantled phenotype. It should be understood however that these examples are given only by way of illustration of the invention and do not constitute in any way a limitation thereof. EXAMPLES

General methods

Plant material

Oil palm tissue cultures were established and maintained as previously described by PANNETIER et al .

(Oleagineux, 36, 119-122, 1981) . Two different clonal lines,

LAB146 and LAB147, were used for differential display analysis. The LAB146 culture was obtained by direct cloning of a seed-derived palm and may therefore be assumed to carry little or no mantled abormality (i.e. it should generate either a low percentage of mantled regenerants or only normal clonal progeny) . The LAB147 culture was obtained by recloning a mantled tissue culture-derived palm and can therefore be assumed to produce 100% abnormal regenerants as illustrated by RIVAL et al . , (Plant Tiss. Cult. Biotechnol., 3, 74-83, 1996) . For northern analysis, a range of cultures was used. Tissues were harvested corresponding to 3 developmental stages in the regeneration process: nodular callus, somatic embryos and 1 cm shoot apex-containing segments excised from leafy shoots. Inflorescence material was obtained from of seed-derived and tissue culture-regenerated oil palms grown in Cδte d'lvoire and Malaysia.^' Root and leaf material was harvested from seed-derived greenhouse plants. For DNA extraction, leaf material from adult palms of several different genotypes (see below) was used. Extraction and analysis of ENA and DNA

All standard cloning procedures used in this study were carried out essentially as described by (SAMBROOK et al . , Molecular Cloning: A laboratory manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, USA, 1989) unless otherwise indicated. Total RNA was extracted as described previously by CORRE et al . , (L. Plant Sci . , 117, 139-149, 1996) . RNA gel electrophoresis and northern transfer were carried out using a NorthernMax-Gly® kit (AMBION CORPORATION) . Membranes were hybridised with either ³ P- radiolabelled RNA probes synthesised using a Strip-EZ® RNA probe kit (AMBION CORPORATION) or ³ P-radiolabelled DNA probes obtained using the random priming method (FEINBERG and VOGELSTEIN, Analytical Biochemistry, 132, 6-13, 1983) . Genomic DNA was extracted as previously described by RIVAL et al . , (Plant Breeding, 117, 73-76, 1998) and analysed by Southern blotting and hybridisation using standard techniques.

Differential display analysis

Differential display analysis was carried out using the primers and PCR conditions described by MALHOTRA et al . (Nucl. Acids Res., 26, 854-856, 1998) with [ -³³P]dATP as the radiolabel. After denaturing polyacrylamide gel electrophoresis, differential bands were excised and re- amplified by conventional PCR using the same primers as were used for the differential display amplifications. Re- amplified DNAs were blunt end cloned into the EcoRM site of the pBluescript SK- phagemid® (STRATAGENE) . Several clones were sequenced for each marker re-amplification reaction and when more than one cDNA sequence was found to be cloned for a given marker, each cDNA was tested separately.

Construction and screening of cDNA library A cDNA library was constructed from 1 cm shoot apex-containing segments excised from leafy shoots of the LAB147 culture grown for 3 weeks on medium containing 10-5 M benzylaminopurine (BAP) . Screening was carried out by plaque hybridisation at 60°C in a buffer containing 5 x Denhardt's solution, 6xSSC, 4 μg/ml sheared salmon sperm DNA and 0.5% SDS. EXAMPLE 1: CHARACTERISATION BY DIFFERENTIAL DISPLAY OF A CDNA FRAGMENT OVEREXPRESSED IN MΑNTLED-BEΑRT G TISSUE CULTURES

Differences in mRNA accumulation between shoot apex-containing segments of the LAB146 (normal seed palm- derived) and LAB147 (abnormal clone-derived) cultures were studied using differential display, enabling the identification of differential bands specific to normal or abnormal material which were subsequently excised, re- amplified and cloned. One of the abnormal-specific cDNA fragments obtained, provisionally named m5B, was shown by Northern hybridisation to correspond to an mRNA of ca. 0.6 kb which accumulates at higher levels in LAB147 shoot apex-containing segments than in those obtained from the LAB146 culture, as shown in Figure 1.

Figure 1 shows a northern hybridisation analysis of transcript accumulation using a ³²P-radiolabelled RNA probe synthesised from the m5B differential display clone containing part of the EGADl cDNA sequence. The m5B (EGADl ) probe was hybridised to total RNA extracted from oil palm tissue cultures and organs of intact plants (upper panel) . As a reference, a control hybridisation to oil palm ³²P- radiolabelled 25S ribosomal DNA probe was also performed (lower panel) . Two different tissue cultures were examined: LAB146, a normal seed palm-derived culture (labelled "SPDC") and LAB147, an abnormal (mantled) clone-derived culture (labelled "ACDC"). Other abbreviations are as follows:

- INFLO, spikelets of female inflorescences;

- N, normal inflorescence (25 cm in length) ; - A, abnormal inflorescence (30 cm in length) .

In a preliminary characterisation of the tissue specificity of the m5B mRNA, it was found that the difference in accumulation of the 0.6 kb transcript between the LAB146 (normal) and LAB147 (abnormal) cultures was even more marked for calli than for shoot apex-containing segments. Little or no difference in signal intensity was observed for somatic embryos.

In whole plants, the 0.6 kb transcript was found to be present in both normal and abnormal female inflorescences (25 cm and 30 cm respectively in length) harvested from clonal palms, similar levels being observed in each case.

No m5B-specific signal was observed for leaves or roots of intact seedling plants, although a longer exposure of the autoradiograph shown produced a faint signal for roots .

Overall, it can be concluded from the Northern hybridisation shown in Figure 1 that transcripts corresponding to the m5B cDNA accumulate principally in inflorescence tissues in the intact plant, as well as in tissue cultures in an apparently man le -dependent fashion.

EXAMPLE 2: ISOLATION AND CHARACTERISATION OF THE FULL LENGTH EGADl CDNA The cDNA insert in the m5B clone was used as a probe to screen an oil palm cDNA library prepared from shoot apex-containing segments of leafy shoots grown on BAP- containing medium, allowing the purification and sequencing of 6 positive clones containing the previously determined m5B nucleotide sequence. A database search revealed that each of the cDNAs thus obtained codes for a cysteine-rich polypeptide sharing close similarities with the plant defensin (or γ-thionin) proteins (TERRAS et al . , Plant Cell, 7, 573-588, 1995) . Although slight variations in size were observed between the cloned cDNAs, only one (m5B-15) displayed a divergent nucleotide sequence, the others all being base identical. Since the m5B-15 cDNA diverges from the other 5 cDNAs at only 3 nucleotide positions, we believe that it might represent a different allele of the same gene locus. This hypothesis is consistent with our genomic Southern data

(see Example 3 below) . The longest cloned cDNA obtained, m5B-7, was selected for use in subsequent experiments and renamed EGADl . The nucleotide sequence of the 535 bp EGADl cDNA and the deduced sequence of the polypeptide for which it codes are represented respectively as SEQ ID NO: 1 and SEQ ID NO: 2 and are also shown in Figure 2.

Figure 2 also shows the nucleotide sequence of the EGADl (m5B-7) cDNA and the deduced sequence of the encoded polypeptide. The position of the putative signal peptide sequence is indicated by underlining. The position of the putative translation termination codon is denoted by an asterisk.

EGADl codes for a 77 amino acid polypeptide of predicted size 5.3 kD. The EGADl cDNA sequence shown in Figure 2 is assumed to contain a full length coding region, since it includes a putative translation initiation codon which is conserved amongst plant defensins and which is preceded in the same reading frame by a TAG stop codon 12 bp further upstream. The polypeptide encoded by the EGADl gene contains a putative signal peptide sequence of 30 amino acids which is likely to be responsible for targeting the nascent polypeptide into the secretory pathway. In order to examine amino acid sequence conservation between the EGADl precursor and those of other defensin/γ-thionin proteins, a CLUSTAL alignment was carried out. The EGADl precursor sequence was aligned, using the CLUSTAL W program (THOMPSON et al . , Nucl . Acids Res., 22, 4673-4680, 1994) with those of its 3 closest published relatives, namely the products of the genes PPT from Petunia infla ta (KARUNANANDAA et al . , Plant Mol. Biol . , 26, 459-464, 1994), Jl -1 from Capsicum annuum (MEYER et al . , Plant Physiol., 112, 615-622, 1996) and P322 from potato (STIEKEMA et al . , Plant Mol. Biol., 11, 255-269, 1988).

Figure 3 shows the alignment of the deduced EGADl protein sequence with those encoded by the genes PPT from

Petunia infla ta (SEQ ID NO: 3), jl-1 from Capsicum annuum

(SEQ ID NO: 5) and P322 from potato (SEQ ID NO: 4). Asterisks indicate residues which are absolutely conserved in all 4 polypeptide sequences, whilst single and double dots respectively denote conservations within "weaker" or "stronger" groups as defined within the CLUSTAL program. The position of the putative translation termination codon is indicated by an asterisk.

The closest identified relative of EGADl is PPT, the two respective sequences sharing 63.6% identical residues. It is interesting to note that the positions of the 8 cysteine residues are absolutely conserved between all 4 polypeptides, strongly suggesting that they all share the same secondary structure. Defensins are thought to play a role in pathogen defence and in some cases have been shown to exert an antifungal action, probably brought about by electrostatic interaction with hyphal cell membranes (THEVISSEN et al . , J. Biol. Chem. 271, 15018-15025,- 1996). EXAMPLE 3: ESTIMATION OF THE NUMBER OF EG&D1-LIKE GENE LOCI PRESENT IN THE OIL PALM GENOME

Oil palm genomic DNA was extracted from leaves of 3 different adult oil palms, X, Y and E, of different genotype in each case (NB palms X and E were used to obtain the tissue cultures XI and LAB146 respectively) . DNAs were digested with 3 different restriction enzymes, Southern blotted and hybridised to the EGADl cDNA.

Figure 4 shows a Southern hybridisation of a ³²P- radiolabelled EGADl cDNA probe . to oil palm genomic DNA digests from the three genotypically different oil palms, X, Y and E, . The restriction enzymes used and size marker migration positions are indicated.

These results suggest that the EGADl gene occurs as a single copy per haploid genome. All 3 restriction enzymes used (none of which cuts the EGADl cDNA) were found to produce only one hybridising fragment, except in the case of EcoRI when used with DNA of the Y genotype; in this case, a doublet was observed. One possible explanation for the latter result is that it might be caused by a difference in restriction pattern between allelic regions in or bordering the EGADl gene locus in the Y genome.

EXAMPLE 4: INVESTIGATION OF THE aNTI-ED-DEPENDENT EXPRESSION OF EGADl IN OTHER OIL PALM GENOTYPES AND CULTURES

Since the northern hybridisation data shown in Figure 1 revealed a strong differential accumulation of EGADl transcripts at the callus stage of tissue culture regeneration, we investigated this phenomenon further. The N and A cultures used for the differential display and preliminary expression analysis were not of identical genotype; thus it was important to check that the difference in transcript levels seen was not simply attributable to differences in genetic background. We therefore investigated EGADl transcript accumulation in genotypically identical cultures differing only in their mantled status, namely the cultures XI (normal) , X2 (intermediate) and X3 (abnormal) . These cultures were initiated from respectively a normal seed-derived palm, a normal regenerant palm previously cloned from the seed-derived palm and an abnormal regenerant palm again previously cloned from the same original palm. The XI, X2 and X3 cultures were all initiated at the same time, so as to eliminate any possible effects due to culture age. In terms of abnormality, the X2 culture can be considered as representing an intermediate situation in that it has been initiated from a normal palm, but consists of cells which have undergone an extra round of tissue culture compared with the XI culture. In the same experiment, in order to check the reproducibility of EGADl expression between different cultures obtained from a given starting material, we analysed callus lines N' (LAB148) and A' (LAB149), which were respectively initiated from the same palms as the LAB146 and LAB147 cultures. Figure 6 shows the northern hybridisation profiles obtained with each callus line using the EGADl probe. The hybridisation profiles of the N' and A' samples are consistent with those shown in Figure 1 for the N and A lines, thus confirming the reproducibility of these results between cloning experiments and providing further evidence that the differential EGADl gene expression patterns observed may be associated with the mantled abnormality. This hypothesis is further strengthened by the hybridisation profile observed for the XI, X2 and X3 cultures, which differ in their mantled status within a common genotypic background. As expected, the X3 (abnormal) culture produces a dramatically stronger signal than the XI (normal) culture. It is moreover interesting to note that the X2 (intermediate) culture produces a signal greater than that of XI, but weaker than that of X3, presumably reflecting the extra cycle of tissue culture "history" which it carries compared with the XI line.

The above data confirm that EGADl gene expression is affected by epigenetic factors which prevail in tissue culture, resulting in increased transcript accumulation in abnormal clonal lines at the callus stage. The reproducibility of EGADl gene expression profiles between cultures established at different times from the same palm has been demonstrated. Most importantly, studies performed on the genotypically homogeneous X1/X2/X3 cultures shows that EGADl gene expression provides a sensitive marker for the presence of the man tled abnormality at the callus stage.

Claims

CLAIMS 1) An isolated polypeptide of the defensin family, hereinafter referred as EGADl, having at least 70% sequence identity with the polypeptide of SEQ ID NO: 2. 5 2) A fragment of an EGADl polypeptide of claim 1, comprising at least 5 contiguous amino-acids of said polypeptide.

3) An isolated polynucleotide selected among : a) a polynucleotide encoding an EGADl polypeptide L0 of claim 1; b) a fragment of a polynucleotide a) comprising at least 10 contiguous nucleotides ; c) a polynucleotide complementary of a polynucleotide a) or b) above;

.5 d) a polynucleotide hybridising selectively under stringent conditions with a polynucleotide a) b) or c) above.

4) A polynucleotide of claim 3 selected among : a) the cDNA of SEQ ID NO:l; b) a polynucleotide comprising at least 10 '0 contiguous nucleotides of SEQ ID NO: 1; c) a polynucleotide complementary of a polynucleotide a) or b) above; d) a polynucleotide hybridising selectively under stringent conditions with a polynucleotide a) b) or c) above.

5 5) An antibody directed against a polypeptide of any of claims 1 or 2.

6) The use of a polypeptide of any of claims 1 or 2 or of a polynucleotide of any of claims 3 or 4, or of an antibody of claim 5, for detecting the mantled phenotype in an

0 oil palm tissue culture.

7) A method for detecting the mantled abnormality in an oil palm tissue culture, wherein said method comprises detecting the overexpression of the EGADl gene, encoding an EGADl polypeptide of claim 1, in said culture.

5 8) A method of claim 7, wherein the overexpression of the EGADl gene is detected in an oil palm tissue culture at the callus stage. 9) A method of any of claims 7 or 8, wherein the overexpression of the EGADl gene is detected by evaluating the amount of EGADl transcripts.