2012 Annual Report
1a.Objectives (from AD-416):
Effective genetic strategies are needed to address major crop loss due to pathogen disease and provide alternatives to current chemically based crop protection strategies that impose human health and environmental risks. The long-term objective of this project is to gain an improved understanding of the structure, function and evolution of key components of the plant innate immune system for strategic deployment of key components of this highly regulated system to reduce the threat of crop loss by pathogens. Our specific objectives are to use evolutionary models and molecular-genetic strategies to identify durable pathogen resistance genes, to use genomics and genetics to identify and understand functional and regulatory components of the plant innate immune system, and to use comparative structural and functional genomics to understand the mechanism of induction and function of cellular and systemic events that constitute effective induced defense responses and plant innate immunity. Identification of functional and regulatory components of the innate immune system, and understanding the process of induction of defense responses will provide the conceptual base for deploying strategies for broad-spectrum resistance.
1b.Approach (from AD-416):
1. Use an evolutionary model developed by this project, in combination with positional cloning, to isolate the PVX resistance gene, Nb. Develop DNA markers for fine genetic mapping of the virus disease resistance locus Nb. Develop PCR methods to isolate candidate disease resistance locus Nb. Perform complementation tests for functional confirmation of Nb isolation. Test performance of Nb in potato cultivars.
2. Identify and analyze transposable elements in the Solanaceae and assess their impact on gene expression in plant innate immunity. Perform comparative analysis of Solanaceae genomic sequences to identify repetitive transposable element-derived sequences inserted within R-gene hotspots and other genes and intergenic regions for further exploration of their role in structural diversity and genome evolution. Explore transcription of MITE-derived candidate regulatory sequences by mining Solanaceae EST databases for the presence of MITE sequences. Explore functional role of candidate MITE sequences in biotic stress by: identifying MITE-derived small RNAs, studying their biogenesis and comparing expression of MITE small RNAs and their candidate targets in biotically challenged and unchallenged wild-type and RNAi plant lines.
3. Understand induced defense responses associated with plant innate immunity by studying host and pathogen effector interaction with cell proteins.
This is the final report for the project 5335-22000-007-00D, which terminated in March, 2012. It has been replaced by project 5335-22000-008-00D Manipulation of Plant Disease Resistance Genes for Improved Crop Protection. For additional information see the report for project 5335-22000-008-00D. We made significant progress related to resources for identification of plant disease resistance regulatory sequences, genes and signaling components for crop protection strategies 5 years of the project. To identify and test regulatory sequences involved in R-gene evolution and expression we established small RNA and transcriptome databases, bioinformatic tools and transgenic lines with reduced small RNA production. Our previous investigations to understand the evolution of new R-genes to combat emerging pathogen strains showed that plant genomes harbor high-copy numbers of tandemly arrayed, fast evolving R-gene sequences as well as abundant and small, gene associated transposons. We used genetic and comparative genomic analyses to show that R-gene and gene associated transposons generated small interfering RNAs required for gene silencing and we proposed that silencing played a role in Solanaceae R-genes evolution and expression. The resources we established were key to the identification of novel families of microRNAs and secondary siRNAs that silence and regulate R-genes. The impact of the research was the discovery of a conserved two-part silencing system mechanism that regulates expression of high-copy R-gene families and thus enables retention of high copies for their continuing diversification and evolution. We also made progress on identification of a new resistance gene, Nb for potato virus X resistance, using an evolutionary model. The isolation of Nb is important because it demonstrated the importance of understanding the evolutionary characteristics of an R-gene for its isolation and provides a new gene in the arsenal for combating significant pathogen diseases in the important crop potato. Progress was also made in identification of signaling components of plant disease resistance. To identify signaling components in plant immunity we determined that S-nitrosoglutathione reductase (GSNOR1) plays a role in pathogen responses including N-mediated TMV resistance. GSNOR1 mitigates nitrosative stress caused by increased nitric oxide (NO) levels produced during resistance responses. We found that hypersensitive responses triggered by pathogen effector-R-protein interactions in GSNOR1-silenced plants led to uncontrolled spread of the hypersensitive response and to spontaneous cell death in unchallenged plants. The significance of these results is that they confirm that nitric oxide (NO) signaling plays an important role in plant cell death and resistance responses.
A web-server for identification of plant small RNAs and target genes. Plant micro(mi)RNAs and small interfering (si)RNAs target and silence many genes and thus play important roles in multiple and key biological processes. Predictive bioinformatic tools and small (s)RNA, mRNA and genomic DNA sequences are critical resources for identification of miRNAs, siRNAs and their gene targets. However, these resources are not readily accessible to all researchers. Scientists in Albany, California, developed and implemented "SoMART" a public web-Server for miRNA/tasiRNA Analysis Resources and Tools, which is designed to identify miRNA and siRNAs that regulate genes of interest in Solanaceae and other plants. The impact of this work is that researchers can now investigate the role of miRNA regulation of genes that operate in a variety of pathways and optimize expression of target genes for improved biological outcomes in crops.
Silencing mechanisms regulate resistance genes. Catastrophic crop losses caused by pathogen diseases are averted by deployment of disease resistance (R)-genes encoding nucleotide binding–leucine rich repeat (NB-LRR) plant innate immune receptors, which recognize pathogen effectors and trigger cell death resistance responses. Plants have evolved hundreds of R- genes, however, evolving pathogen effectors can evade encode NB-LRR receptor recognition and overcome resistance. Despite the potential serious consequence on agricultural production, mechanisms regulating R-gene expression were not understood. Scientists in Albany, California, discovered that microRNA-mediated gene silencing, known to regulate expression of genes critical to growth and response to the environment, also regulates the many agronomically important R-genes in tomato and potato Solanaceae crops. These findings are important because they show a conserved role for miRNA regulation of plant innate immune receptor gene expression and pathogen resistance and provide the groundwork further discovery of the role of silencing in plant innate immunity. The impact of silencing on R-gene mediated pathogen resistance will inform strategies for optimizing deployment of pathogen resistance genes in crops.