CN108531489B - Rice kernel Smut pathogen effector gene Smut _2965 and application thereof - Google Patents

Abstract

The invention provides a rice Smut pathogen effector gene Smut _2965 and application thereof, wherein the nucleotide sequence of the gene is shown as SEQ ID NO: 1 is shown in the specification; the amino acid sequence of the protein coded by the gene is shown as SEQ ID NO: 2, respectively. The invention can design the pesticide molecular target by cloning and functional analysis of the rice kernel smut pathogen effector gene; the receptor protein gene of the effector protein in host cells such as rice and the like can be knocked out or mutated to obtain a durable disease-resistant variety; the molecular detection system for the pathogenic variation of the smut bacteria natural population is facilitated to be established, the distribution condition of the smut bacteria effector gene in the field natural population is researched, and the composition and variation characteristics of the microspecies in the smut bacteria population are revealed; and is also helpful for the disease resistance identification of rice varieties and the reasonable layout and rotation of the rice varieties so as to effectively control the occurrence of rice kernel smut germs.

Description

Rice kernel Smut pathogen effector gene Smut _2965 and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a rice Smut pathogen effector gene Smut _2965 and application thereof.

Background

Rice grain smut is a soil-borne fungal disease caused by Tilletia horrida and is distributed in many rice regions of Asia, America and Africa. Ustilago virens has a wide host range and comprises various crops such as rice, corn, sugarcane, sorghum, barley and the like. Ustilago esculenta can infect various crops and is related to effector molecules secreted by the crops, and the molecules can regulate the innate immunity of a host and enhance the infection of the host. It is now generally accepted that these effector molecules are key causative factors in the enhancement of parasitic infections.

The Ustilago oryzae can closely combine with a host structure thereof by secreting effector molecules to control host cells and change the process of host plants. The effector molecule is the major pathogenicity factor in phytopathogen interactions. At present, effector has become a hot spot of research in plant pathogen interaction. ATR1(310 amino acid sequences) and ATR13(150 amino acid sequences) are two effectors with avirulent gene activity of the Arabidopsis pathogen Hyaloperonospora arabidopsis, which induce RPP1-Nd/WsB and RPP13-Nd mediated resistance, respectively. ATR13 has the characteristics of a signal peptide and RXLR motif and, in addition, contains a conserved leucine/isoleucine repeat which is required for recognition by RPP13, and indeed variation in the repeat does not mean that it is required for toxicity. It is noteworthy that at least one of the RPP13 families recognized the allele of ATR13, but the specific mechanism is unclear, suggesting that avirulence and resistance genes have a complex network of mutual recognition. The high variability of ATR1 and ATR13 in h. The role of ATR1 and ATR13 in inhibiting the basal resistance pathway has been established by heterologous expression in the bacterial phytopathogen Pseudomonas syringae DC3000 and transport to host cells. ATR1 and ATR13 both increased toxicity and reduced callose accumulation in arabidopsis thaliana when transported by p.syringae DC3000 into arabidopsis thaliana.

AVR3a is the cytoplasmic RXR effector protein of Phytophthora infestans. AVR3a has dual activity in pathogenic mechanisms, inducing allergic reactions (HR) in plants expressing the R3a gene, but in plants lacking the R3a gene, AVR3a inhibits p.infestans INF1 protein-induced cell death, and these different activities can be separated at the structural level. AVR3a, which had been deleted or mutated at the C-terminal amino acid residue tyrosine 147, retained the R3 a-mediated allergic reaction, but failed to inhibit INF 1-induced cell death. In addition, the two major alleles of AVR3a encode proteins that differ only by two amino acids in the effector domain, but with significant activity differences. Is AVR3a^K80/I103Rather than AVR3a^E80/M103R3a was activated and was a relatively strong inhibitor of INF 1-induced cell death.

In addition to RXLR effectors, studies on p.infestans secreted proteins have identified another cytoplasmic effector family, Crinkler. Crinkler is another large, diverse effector family of oomycetes. High throughput functional screening revealed two cell death-inducing proteins, CRN1 and CRN2, which when expressed systemically in plants cause the leaf-shriveling phenotype. With the availability of p.infestans genomic sequences, studies of the rare regions of genes revealed that CRN gene families co-localize to repeat enrichment regions in addition to the RXLR effector gene, and have dramatically expanded (193 predicted genes) in p.infestans compared to other Phytophthora gene families. The expansion of this gene family has occurred through polygenic replication and genetic internal recombination events that ultimately lead to a large, diverse chimeric effector system. CRN effector is a modular protein characterized by a predicted N-terminal secretory signal peptide, followed by a domain defined by a conserved but not invariant LXLFLAK motif (the main defining feature of CRN proteins). Most CRNs carry a DWL domain following the LXLFLAK domain, ending with the HVLVXXP motif. Haas et al, proposed that recombination between CRNs, especially after the HVLVXXP motif, produced a very diverse C-terminal domain (determined by 36 distinct amino acids in p.infestans). Similar to several RXLR proteins, expression of some CRNs at the C-terminus (and therefore lacking the predicted N-terminal transfer domain) in plant cells induces cell death. However, the relevance of CRN-induced cell death in disease remains unclear.

Yoshida et al found that three candidate effector genes Pex22, Pex31 and Pex33, which were mined from the unique sequence of Magnaporthe grisea lna168, had perfect associations with three avirulent phenotypes, Avr-Pia, Avr-Pik/km/kp and Avr-Pii, respectively. Subsequent genetic transformation experiments demonstrated that the effector Pex22, Pex31, and Pex33 conferred no toxicity to rice culture lines expressing the Pia Pik/km/kp and Pii resistance genes, respectively. Pex22 identified as Avr-Pia was independent by positional cloning. Pex22, Pex31 and Pex33 are all small, 85, 70 and 113 amino acids in length, respectively, contain secretion signals, and are recognized in the cytoplasm of rice cells, which means that they are transported into plant cells during infection. Interestingly Pex22 formed a small family containing four homologues, which is characterized by the presence of two sequence motifs: motif 1 is [ LI ] xAR [ SE ] [ DSE ], and is capable of inhibiting BAX-mediated cell death, similar to the LxAR motif of Avr-Piz-t; motif 2 is [ RK ] CxxCx12H, showing the similarity of the C2H2 zinc finger motif associated with protein-protein interactions.

The transferred effectravrl 567 family from the flax rust Melampsora lini is recognized by direct interaction with the resistance proteins L5, L6 and L7 of flax. This system is particular because the crystal structures of AvrL567-A and AvrL567-D have been elucidated. This allows the detection of the effect of a gene on the basis of its structure of a polymorphic residue associated with a molecular recognition event. Neither protein is close to known structural homologues. Four important highly polymorphic residues are at positions 50, 56, 90 and 96 of AvrL567, which are critical for activating the R protein and are located on the surface of these effector proteins. Interestingly, the distribution of the four residues of AvrL567 spans the entire protein surface, suggesting that the interaction between AvrL567 and the leucine-rich repeat domain of the R protein requires multiple points of attachment with cumulative effects on the overall interaction. This may be important in co-evolution to maintain or develop toxic functions (beneficial for pathogens) while at the same time circumventing detection of the host. The structure of the proteins also reveals two positively charged surface points for binding to DNA, and subsequent in vitro experiments indicate that these proteins bind to nucleic acids. This shows how structural biology facilitates functional studies of the effector when little useful clues are available from the original sequence.

Cloning using the map-based cloning strategy three avirulence genes, AvrLm1, AvrLm6 and AvrLm4-7, were obtained from Leptosphaeria maculons, which encode predicted small secreted proteins from 122 to 205 amino acids, with no similarity to any of the proteins in the public databases. AvrLm6 and AvrLm4-7 contain 6 and 8 cysteine residues, respectively, that may stabilize the AvrLm6 and AvrLm4-7 proteins outside the plastid. However, AvrLm1 has only one cysteine residue, and AvrLm1 is likely to be transported into the host cell given that all the extra-plastid effectors described so far are cysteine-rich proteins. Although we are aware that they have non-toxic activity, the mechanism that AvrLm recognizes, the corresponding Rlm resistance genes and their sites of action remain unclear. The three l.maculans genes have lower GC contents than the effector fungi of most other fungi currently cloned. They are single copy genes, located in AT-rich and transposon-rich heterochromatin-like regions of 60kb (AvrLm4-7), 133kb (AvrLm6), 269kb (AvrLm 1). These effectors are localized in a unique genomic environment and are conditionally independent of the chromosome, subtelomeric region of plasmidium, or the genetic trail of rare regions in p.infestans by fungi, which is thought to accelerate gene evolution and pathogen adaptation to the host.

Filamentous phytopathogens have evolved effector proteins that inhibit protease activity to protect several host hydrolase activities. Host-specific toxins (HSTs) are chemically diverse effector molecules that are produced by phytopathogenic fungi and function as virulence factors. These pathogenicity determinants are only active in host plants susceptible to pathogenic bacteria producing the toxin. Interestingly, several protein HSTs, such as Pyrenophora tritici-repentis and Stagonospora nodorum PtrToxA, SnTox1, SnTox2 and SnTox4 all show an optically dependent pattern of necrosis induction and contribute to the susceptibility of the toxin-sensitive host wheat plant. The corresponding dominant host genes Tsn1, Snn1, Snn2, Snn4, which are necessary for the function of these effectors, are mapped on the chromosome of toxin-sensitive hosts, and the products of these genes are considered to be receptors that interact directly or indirectly with HST. Notably, in HST, which shows different interactions with gene pairs, the receptor molecule is essential rather than resistant in classical avirulence-resistance gene interactions. PtrToxA, the best studied in HST of p.tritierepitans and s.nodorum, has a modular structure with an N-terminal secretion signal, which is cleaved to form the mature protein, followed by an RGD domain and a C-terminal effector domain necessary for host transport. PtrToxA is reported to localize in the chloroplast and to interact with the chloroplast protein ToxABP1, and once it is translocated into the chloroplast, PtrToxA promotes virulence and ultimately induces reactive oxygen species accumulation in a light-dependent manner by interfering with photosystem i and photosystem ii.

Nep 1-like proteins (NLP) are bacterial, fungal and oomycete toxins that induce necrosis in dicots. Despite their diverse distribution among different taxa, NLPs share a common folding feature, namely, a heptapeptide (GHRHDWE) motif and two conserved cysteines, showing structural similarity to the marine-produced anemolysins and cytolytic toxins. The expression of the NLP gene (NPP1 and pinp 1.1) in the semi-biotrophic oomycete pathogens p.sojae and p.infestans is up-regulated during the late humus stage of host infection. These NLPs may contribute to the death of host tissues by their cytolytic activity and thus promote their invasion during the growth of the saprophytic pathogen. Although it is now clear that some NLPs promote virulence as toxins, it is not known that all members of this family have cytolytic activity in their hosts.

It is not clear at present how many of the effector biochemical activities and how they enhance successful reproduction of pathogens. We also have little knowledge of targets for filamentous pathogen effectors, particularly those that migrate into the interior of a host cell.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the rice Smut gene Smut _2965 and the application thereof, the invention separates and clones the effector gene from the rice Smut, analyzes the function of the gene, is beneficial to disclosing the specific interaction and the molecular mechanism of evolution between the rice Smut and rice varieties, and further effectively controls the occurrence of the rice Smut.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

a rice Smut pathogen effector gene Smut _2965 has a nucleotide sequence shown in SEQ ID NO: 1 is shown.

The amino acid sequence of the protein coded by the gene is shown as SEQ ID NO: 2, respectively.

It is understood herein that the skilled person will have NO effect on the activity of the protein, as long as the amino acid sequence of SEQ ID NO: 2, and performing various substitutions, additions and/or deletions of one or more amino acids to obtain amino acid sequences with equivalent functions.

Furthermore, in view of the degeneracy of codons, the gene sequences encoding the proteins can be modified, for example, in the coding regions thereof, without changing the amino acid sequences, or in the non-coding regions thereof, without affecting the expression of the proteins, and thus, the present invention also includes amino acid sequences having the same functions formed by substituting, adding and/or deleting one or more amino acid residues to the gene sequences encoding the proteins.

The invention also provides a vector containing the gene and a host cell containing the vector.

The application of the gene is as follows: (1) designing a pesticide molecular target according to the structure and the function of the gene; (2) the application in improving the rice disease resistance breeding, for example, the gene sequence is connected to any transformation vector containing a fluorescent protein gene, the effector gene and the fluorescent protein gene are covalently introduced into rice or other plant cells by any transformation method, the migration and the positioning of the effector protein which is expressed by being fused with the fluorescent protein in the rice or other plant cells in the plant cells can be observed by using a fluorescence confocal transmission electron microscope, the effector is used as a bait protein to fish out an acceptor protein which is combined with the effector protein in the rice or other plants, the acceptor gene in the rice or other plant cells is knocked out by using a genetic engineering method, or one or more basic groups of the acceptor gene are deleted, added and mutated so as to delete or change the function of the acceptor gene, so that a resistant plant resisting a certain effector is obtained; (3) specific molecular markers are generated according to the gene sequence information, and include but are not limited to SNP (single nucleotide polymorphism), SSR (simple sequence repeat polymorphism), RFLP (restriction endonuclease length polymorphism) and CAP (cut amplified fragment polypeptide), the markers can be used for detecting the dynamic change of physiological races and genetic structures of field rice smut groups and the distribution condition of the effector genes in field natural groups, so that the method is beneficial to the identification of disease resistance of rice varieties and the identification of the races of rice smut germs, and is also beneficial to the reasonable layout and rotation of disease-resistant varieties, and the occurrence of smut is effectively controlled.

The rice kernel Smut pathogen effector gene Smut _2965 and the application thereof provided by the invention have the following beneficial effects:

the invention is helpful to reveal the specific interaction and the molecular mechanism of the evolution between the rice kernel smut germ microspecies and rice varieties through the cloning and the function analysis of the rice kernel smut germ gene. In practice, the molecular target of the novel pesticide can be designed according to the structure and the function of the gene; the receptor protein gene of the effector protein in host cells such as rice and the like can be knocked out or mutated to obtain a durable disease-resistant variety; the molecular detection system for pathogenic variation of the natural population of the smut germs is facilitated to be established, the distribution situation of the smut germs effector genes in the field natural population is researched, and the composition and variation characteristics of the microspecies in the smut germs population are revealed; and is also helpful for the disease resistance identification of rice varieties and the reasonable layout and rotation of the rice varieties so as to effectively control the occurrence of rice kernel smut germs.

Drawings

FIG. 1 shows the PCR amplification detection result of Ustilago oryzae effector gene Smut-2965; wherein M is a molecular weight marker, and is 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp in sequence; lanes 1 and 2 are PCR products of Smut _2965 and Smut _5844, respectively; lane 3 is a negative control (ddH)₂O)。

FIG. 2 shows the result of allergic necrosis reaction caused by transient expression of the effector gene Smut-2965 in tobacco leaves; the left image is the image of Smut _2965 and negative control (no-load) injected for 4 days, and the right image is the image of positive control (mouse apoptosis protein) and negative control (no-load) injected for 4 days.

FIG. 3 is a SDS-PAGE detection result chart of the expression protein of Ustilago oryzae effector gene Smut-2965; wherein, the Lane Marker is a molecular weight Marker which is 250KD, 150KD, 100KD, 75KD, 50KD, 37KD, 25KD and 15KD in sequence; lane 2965-SP shows the signal peptide band, 2965-DMDC shows the induced protein band, and the band of interest appears at 14 kD; lane BAX-DMDC is the positive control mouse apoptosis protein expression band.

Detailed Description

Example 1

1. Isolation and cloning of effector Gene

Inoculating a rice smut bacterial strain separated from a rice smut sample to a PSA solid culture medium for activation, selecting a single colony in a 50mL PSA liquid culture medium after 5 days, culturing for 5 days at 28 ℃ and 200r/min, filtering and collecting hyphae by four layers of gauze, grinding by liquid nitrogen to extract total RNA, and carrying out reverse transcription by using oligodT as a primer to obtain cDNA.

Primers were designed based on the predicted sequence, and the primer sequences were as follows:

a forward primer:

5′-GACCTCGACTCTAGAGGATCCATGAAGTTTGCCACCCTCGC-3′(SEQ ID NO：3)；

reverse primer:

5′-GTCCTTGTAGTCAGAAGGCCTGTAAGAGCACTTAAGGAAGCTGCTG-3′(SEQ ID NO：4)。

PCR amplification was performed using cDNA as template, PCR reaction system (50. mu.L): 2 μ L of cDNA template, 2 μ L of forward and reverse primers, 10 μ L of Fastpfu Fly Buffer, 5 μ L of 2.5mM dNTPs, 1 μ L of Fastpfu Fly Hi-Fi enzyme, ddH₂O28 mu L; the PCR amplification procedure was: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 50s, annealing at 55 ℃ for 50s, and extension at 72 ℃ for 1min for 35 cycles; stretching for 10min at 72 ℃. And (3) carrying out electrophoretic detection on the PCR amplification product, wherein the obtained target gene has no impurity band in electrophoretic detection as shown in figure 1.

2. Construction and transformation of prokaryotic expression vector of effector gene

The target gene fragment amplified by PCR is recovered by an agarose gel DNA recovery kit (OMEGA, USA), the target gene is connected to an expression vector pEASY-E1 according to the description of pEASY-Blunt E1kit, 2 mu L of the connection product is taken to be mixed with 50 mu L of DH5 alpha competent cells (CW0808S, CWBIO, Beijing), the mixture is coated on YEP solid culture medium containing kanamycin (50mg/L) and rifampicin (50mg/L) for selective screening to obtain a recombinant plasmid, and after sequencing, the gene Smut-2965 is completely identical to the predicted sequence, and the gene sequence is shown as SEQ ID NO: 1 is shown. After GV3101 competent cells were electrically transformed and rifampicin and kanamycin resistance were screened, Agrobacterium strains carrying transient expression vectors were obtained.

3. Transient expression in Nicotiana benthamiana leaves

Marking and separating the agrobacterium carrying the target gene to obtain single clone, picking the single clone and placing it in the plant containing the target geneFuping (50mg/L) and kanamycin (50mg/L) at 28 ℃ for 16h, centrifuging, collecting the thallus, MES resuspension [10mM MES (pH5.6),10mM MgCl₂And 150. mu.M acetosyringone]The cells were resuspended and the OD600 was adjusted to 0.5. Then, the tobacco leaves were cultured at room temperature in the dark for 3 hours, injected with a syringe, and observed, and the results are shown in FIG. 2.

As can be seen from FIG. 2, the negative control (Agrobacterium liquid without any expression vector) does not cause the necrosis reaction of the nicotiana benthamiana leaves after injection, and the positive control (Agrobacterium liquid with mouse apoptosis protein BAX) and the Agrobacterium liquid with the target gene Smut _2965 cause the obvious necrosis reaction after injection into the nicotiana benthamiana leaves 4d, which indicates that the Smut _2965 gene can generate the pathogenic reaction.

4. Transient expression validation in Nicotiana benthamiana leaves

200g of Nicotiana benthamiana leaves injected for 72-96h are ground into powder by liquid nitrogen, added into a sterile 2mL EP tube, added with 500uL of protein for extraction buffer, vortexed for 60s and frozen for 30 min. 14000r/min, centrifuging at 4 ℃ for 10min, taking the supernatant, adding a loading buffer, carrying out water bath at 100 ℃ for 5-10min, centrifuging at 12000r/min for 1min, and taking 10uL for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis. The results are shown in FIG. 3, wherein lanes 2965-DMDC represent the expression protein band of effector smut _2965, and lanes BAX-DMDC represent the expression protein band of apoptosis protein of positive control mice.

5. Application of effector gene Smut _2965

By utilizing the sequence information of the Smut _2965 gene provided by the invention, a molecular target of a novel pesticide is designed according to the structure and the function of the gene; the opportunity of the gene protein product in the receptor protein gene or the signal path of the rice is silenced or knocked out so as to cultivate disease-resistant varieties; the application of the molecular marker generated according to the gene sequence in monitoring the population of the rice smut in the field; and guiding the application of the disease-resistant variety in reasonable layout according to the monitoring result.

Sequence listing

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gtcgagtact ctgactacgg ctacgctccc cacctaccct caccagtaca acctactgct 180

ccgacaccaa gttctacgag tacccctgac cagcaacggc tacactggcg gctctcccgg 240

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gtccttgtag tcagaaggcc tgtaagagca cttaaggaag ctgctg 46

Claims (2)

1. A protein coded by a rice Smut gene Smut _2965 of Ustilago oryzae is disclosed, and the amino acid sequence of the protein is shown as SEQ ID NO: 2, respectively.

2. The application of the protein coded by the rice Smut gene Smut _2965 of claim 1 in designing a pesticide molecular target.

Images