Source: KANSAS STATE UNIV submitted to
FUNCTIONAL ANALYSIS OF THE AVR-PITA RICE BLAST AVIRULENCE PROTEIN IN PATHOGENICITY AND HOST SPECIFICITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0193381
Grant No.
2002-35319-12567
Project No.
KS9717
Proposal No.
2002-02531
Multistate No.
(N/A)
Program Code
51.8
Project Start Date
Sep 1, 2002
Project End Date
Aug 31, 2006
Grant Year
2002
Project Director
Valent, B.
Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
PLANT PATHOLOGY
Non Technical Summary
Plants possess a surveillance - defense system in which resistance genes recognize signal molecules produced by corresponding pathogen avirulence (AVR) genes. This project will determine the biochemical role that one fungal AVR gene plays during infection of rice and in sometimes triggering resistance. Such an understanding is a first step toward producing durable disease resistance in crops
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20115301060100%
Goals / Objectives
The overall goal of this project is to understand the biochemical role of a fungal avirulence protein in promoting pathogenicity and disease development, and in sometimes inducing the hypersensitive resistance response in rice. Specific objectives are: (1) Optimize an in vitro expression system for producing recombinant AVR-Pita protein using the yeast Pichia pastoris. (2) Demonstrate protease activity and substrate specificity by recombinant AVR-Pita protein using a range of protein and peptide substrates, and directly test if proteolytic cleavage of the Pi-ta resistance protein by AVR-Pita might play a role in the recognition response. (3) Purify and analyze AVR-Pita protein from infected plant tissue to confirm its structure in vivo, inside the developing rice blast lesion, and determine the natural substrate for AVR-Pita in the host plant.
Project Methods
This award will investigate the hypothesis that proteolytic events catalyzed by the AVR-Pita avirulence protein play a critical role both early and late in the rice blast disease cycle. Direct biochemical demonstration of protease activity will provide an important test of this hypothesis and subsequently enable detailed analysis and manipulation of molecular mechanisms that trigger the resistance response. We will produce large quantities of recombinant AVR-Pita protein using a yeast expression system, and directly test the recombinant protein for protease activity using a range of protein and peptide substrates. We will also produce recombinant Pi-ta rice resistance protein for use as a substrate to directly test if the putative AVR-Pita protease might degrade the Pi-ta protein during the early recognition process. We will purify and analyze AVR-Pita protein from infected plant tissue to confirm its structure in vivo, inside the developing rice blast lesion. Experiments to determine the natural substrate for AVR-Pita in the host plant include comparative 2-D gel electrophoresis of plant tissue infected by isogenic strains of fungus containing or lacking AVR-Pita. Differences in the protein profiles with and without AVR-Pita will be examined to define which plant proteins are degraded by the AVR-Pita protease

Progress 09/01/02 to 08/31/06

Outputs
We previously cloned the fungal avirulence AVR-Pita gene and its corresponding resistance (R) gene, Pi-ta, from rice, and demonstrated that AVR-Pita protein interacts directly with the Pi-ta protein inside rice cells to trigger resistance. This proposal addressed the hypothesis that proteolytic events catalyzed by AVR-Pita protein play a critical role in promoting disease development, and in inducing hypersensitive resistance in rice containing Pi-ta. We developed an efficient protein expression system for producing AVR-Pita protein in the yeast Pichia pastoris. Although we detected low levels of AVR-Pita protease activity using zymogram gels with blue-stained casein incorporated as a substrate, we were unable to develop a reproducible assay for AVR-Pita protease using a series of standard protein or fluorogenic peptide substrates. These results suggested that the AVR-Pita protease has a highly specific substrate inside its host plant cells. At this point, understanding the dual roles of AVR-Pita in rice infection required us to move to in planta studies. To detect AVR-Pita and Pi-ta proteins in planta, we developed methods for assessment of infection density and of infection quality relative to disease in the field, and we defined assay conditions that enriched for invaded rice cells at the point when AVR-Pita and Pi-ta are known to interact, early during biotrophic blast invasion. We then performed experiments to visualize AVR-Pita inside rice cells invaded with fungus producing AVR-Pita::GFP fusion proteins, as a first attempt to directly demonstrate AVR-Pita delivery by the fungus into the rice cell cytoplasm. These experiments led to the exciting discovery of a novel membrane-rich structure at the interface between the blast fungal wall and the rice cell cytoplasm. We demonstrated that AVR-Pita and additional avirulence proteins accumulate in these structures. These previously unknown structures appear to play a key role during development of rice blast disease. Our current working hypothesis is that they are involved in secretion of avirulence proteins/effectors inside living rice cells. A publication describing this exciting new structure will credit this grant for support leading to this discovery.

Impacts
These studies have led to exciting new insights on the biology of biotrophic blast invasion, the critical stage when blast AVR genes function to promote disease. A future research challenge lies in understanding the biology of effector/avirulence (AVR) proteins that fungal pathogens deliver inside living plant cells in order to control the plant's cellular processes. These same effector/AVR proteins are the targets for recognition by the plant's powerful surveillance-defense system in which resistance (R) genes recognize signal molecules produced by corresponding pathogen AVR genes. Although this resistance is powerful when triggered, highly variable pathogens like Magnaporthe grisea have the ability to evade detection by modifying the AVR gene-encoded signal. We discovered that effector/AVR proteins accumulate within a novel interfacial structure between the fungus and the rice cytoplasm during biotrophic blast invasion. Future studies to understand the role for these novel structures during blast disease will lead to insights on roles for fungal effector proteins and on how they are secreted inside living plant cells. The AVR-Pita protease triggers resistance in rice with the Pi-ta resistance gene, which has direct relevance for controlling rice blast in the US. In addition, understanding fungal effector biology has potential to impact disease control strategies for other harder to study plant diseases such as the rusts and mildews, which also employ a biotrophic invasion strategy in destroying agricultural crops.

Publications

  • Berruyer, R., S. Poussier, P. Kankanala, G. Mosquera, and B. Valent. 2006. Quantitative and qualitative influence of inoculation methods on in planta growth of rice blast fungus. Phytopathology, 96:346-355.


Progress 01/01/05 to 12/31/05

Outputs
Plants possess a surveillance; defense system in which resistance (R) genes recognize signal molecules produced by corresponding pathogen avirulence (AVR) genes. Although this resistance is powerful when triggered, highly variable pathogens like Magnaporthe grisea have the ability to evade detection by modifying the AVR gene; encoded signal. We previously cloned the fungal AVR-Pita gene and its corresponding R gene, Pita, from rice, and demonstrated that AVR-Pita protein binds directly to Pita protein in triggering resistance. This proposal addressed AVR-Pitas dual role as both a fungal virulence factor in promoting disease and an avirulence factor triggering Pita mediated resistance. Our working hypothesis was that proteolytic events catalyzed by AVR-Pita protein play a critical role in promoting disease development, and in inducing hypersensitive resistance in rice containing Pita. One goal was to develop the yeast Pichia pastoris as an efficient protein expression system for producing AVR-Pita protein for biochemical analysis. We developed yeast strains that secrete AVR-Pita protein in high quantities into the culture medium. We detected low levels of AVR-Pita protease activity using zymogram gels with blue-stained casein incorporated as a substrate. However, we have been unable to develop a reproducible assay for AVR-Pita protease using a series of standard protein or fluorogenic peptide substrates. Our results indicate that the AVR-Pita protease has a highly specific substrate. The possibility remains that Pita itself is a substrate for AVR-Pita. However, we were unable to express Pita, as an intact protein or in separate domains, in P. pastoris, and we have not detected AVR-Pita-mediated proteolytic cleavage of the small quantities of Pi-ta proteins produced in E. coli. At this point, understanding the dual roles of AVR-Pita in rice infection required us to move to in planta studies. To detect AVR-Pita and Pi-ta proteins in planta, we needed to enrich for fungus-plant interaction sites in infected plant tissues at the early time points of interest, 24 to 36 hai. We developed methods for assessment of infection density and of infection quality relative to disease in the field1. The rice leaf sheath system we developed for coupling molecular analyses and live cell microscopy of biotrophic blast invasion has provided novel insights on rice blast biology2.

Impacts
These studies have led to exciting new insights on the biology of biotrophic blast invasion, the critical stage when AVR genes function to determine if disease will occur. Understanding the dual resistance and pathogenicity functions of AVR genes is a step toward producing durable disease resistance. The AVR-Pita protease triggers resistance in rice with the Pita resistance gene, which has direct relevance for controlling rice blast in the US.

Publications

  • No publications reported this period


Progress 01/01/04 to 12/31/04

Outputs
The overall goal of this project is to understand the biochemical role of a fungal avirulence protein in promoting pathogenicity and disease development, and in inducing the hypersensitive resistance response in rice containing the corresponding resistance gene. Specific objectives and progress toward meeting these objectives are: (1) Optimize an in vitro expression system for producing recombinant AVR-Pita protein using the yeast Pichia pastoris: This objective has been accomplished as previously described. (2) Demonstrate protease activity and substrate specificity by recombinant AVR-Pita protein using a range of protein and peptide substrates, and directly test if proteolytic cleavage of the Pi-ta resistance protein by AVR-Pita might play a role in the recognition response: We previously reported AVR-Pita protease activity using zymogram gels with blue-stained casein incorporated as a substrate. We predict that AVR-Pita cleaves after 2 or 3 basic amino acid residues based on the cleavage site between the propeptide sequence and the mature enzyme. We are testing for cleavage of fluorogenic peptide substrates to determine the protease cleavage specificity, but we have not yet seen activity with these substrates. Interestingly, the Pi-ta resistance protein, unlike other cloned resistance gene products, contains an abundance of the predicted cleavage sequences. We are expressing Pi-ta protein in order to directly test its susceptibility to proteolytic cleavage by AVR-Pita. However, we have not been able to detect expression of the full length Pi-ta protein in P. pastoris. We are now attempting to express Pi-ta protein domains in yeast and to express the intact protein in E. coli. (3) Purify and analyze AVR-Pita protein from infected plant tissue to confirm its structure in vivo, inside the developing rice blast lesion, and determine the natural substrate for AVR-Pita in the host plant: We have developed a leaf sheath infection assay that allows enrichment of developing infection sites at the early time points. This assay allows us to easily detect the AVR-Pita mRNA using RT-PCR, so that we know the gene is transcribed in these tissues. We have developed protocols to isolate total protein extracts from this infected rice tissue. However, we have not yet been able to identify AVR-Pita protein within these extracts using our AVR-Pita antibodies.

Impacts
The AVR-Pita protease triggers resistance in rice with the Pi-ta resistance gene, which has direct relevance for controlling rice blast in the US. This project will produce a molecular understanding of how resistance is triggered by interaction of these fungal and plant proteins, and of AVR-Pitas role in helping the fungus cause disease. Understanding the dual resistance and pathogenicity functions of AVR genes is a step toward producing durable disease resistance.

Publications

  • No publications reported this period


Progress 01/01/03 to 12/31/03

Outputs
The overall goal of this project is to understand the biochemical role of a fungal avirulence protein in promoting pathogenicity and disease development, and in inducing the hypersensitive resistance response in rice containing the corresponding resistance gene. Specific objectives and progress toward meeting these objectives are: (1) Optimize an in vitro expression system for producing recombinant AVR-Pita protein using the yeast Pichia pastoris: This objective has been accomplished. We have expressed AVR-Pita protein in P. pastoris using a secretion expression vector. The AVR-Pita protein is secreted into the yeast culture medium at high enough quantities that it appears as a major band in a coomassie-stained gel. N-terminal peptide sequencing of this P. pastoris-produced protein has shown that the prepro-protein is processed to a mature enzyme as we predicted. The abundant source of AVR-Pita protein allowed us to demonstrate that the AVR-Pita antibody produced previously against recombinant protein from E. coli is high quality and meets our needs for future experiments. (2) Demonstrate protease activity and substrate specificity by recombinant AVR-Pita protein using a range of protein and peptide substrates, and directly test if proteolytic cleavage of the Pi-ta resistance protein by AVR-Pita might play a role in the recognition response: We have demonstrated AVR-Pita protease activity using zymogram gels with blue-stained casein incorporated as a substrate. Protease activity is visualized as a clear band against a dark background. We are currently purifying AVR-Pita protein for use in enzyme assays with fluorogenic peptide substrates to determine the protease cleavage specificity. We are building expression vectors to produce Pi-ta protein in the yeast system in order to test its susceptibility to proteolytic cleavage by AVR-Pita. (3) Purify and analyze AVR-Pita protein from infected plant tissue to confirm its structure in vivo, inside the developing rice blast lesion, and determine the natural substrate for AVR-Pita in the host plant: We have developed protocols to isolate total protein extracts from infected rice tissue and we are working to identify AVR-Pita protein within these extracts using our AVR-Pita antibodies.

Impacts
Demonstration of AVR-Pita protease activity allows a direct test of the hypothesis that proteolytic events catalyzed by this avirulence protein play a critical role both early and late in the rice blast disease cycle. Understanding dual functions of fungal AVR genes in triggering resistance and in promoting pathogenicity is a step toward producing durable disease resistance in crop plants.

Publications

  • No publications reported this period


Progress 01/01/02 to 12/31/02

Outputs
In the first 4 months of support by this grant, we have initiated the first step towards our goal to identify the role the AVR-Pita avirulence gene plays in rice blast disease. We are working on optimizing the E. coli pET in vitro expression system (Novagen) for expression of the putative AVR-Pita protease and its potential substrate, the LRD domain of rice resistance gene Pi-ta, as outlined in Objective 1. We have improved yields from the bacterial lysis step by adding sonication to our lysis procedure. However, most of the protein is still in the insoluble inclusion body fraction. We have purchased the Pichia pastoris yeast expression system, and we are preparing to subclone AVR-Pita into the Pichia vectors for expression studies.

Impacts
We have taken the first step on a project that will provide understanding of the function of fungal avirulence and plant resistance genes as well as mechanisms of pathogenicity. Such understanding holds promise for designing durable resistance to major plant disease organisms.

Publications

  • No publications reported this period