Source: UNIVERSITY OF CALIFORNIA, BERKELEY submitted to NRP
MOLECULAR STRATEGIES FOR ACHIEVING DURABLE DISEASE RESISTANCE TO BACTERIAL SPOT DISEASE IN TOMATO
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1010249
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2016
Project End Date
Sep 30, 2021
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF CALIFORNIA, BERKELEY
(N/A)
BERKELEY,CA 94720
Performing Department
Plant Biology, Berkeley
Non Technical Summary
A survey will be conducted of the natural field strains isolated from bacterial spot infected tomato plants and the genomes will be sequenced to identify which Xanthomonas Type III effector proteins are conserved in these strains. The identification of conserved effectors will serve as a guide to identify resistance genes that can recognize the conserved effectors. Stacking of multiple disease resistance genes in tomato that recognize the conserved effectors should provide a means to create a more during and sustainable disease resistance to the bacterial spot disease of tomato which will reduce the use of environmentally damaging pesticides. Concomitantly, we will also attempt to construct genome edited tomato plants that will allow less growth and disease symptoms from bacterial infections and ultimately will combine with the stacked resistance genes to construct durable and environmentally friendly bacterial spot disease resistance tomatoes.
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
21240991100100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The objectives of this proposal are the following; 1. Isolate and sequence the genomes of Xanthomonas species from the field that are causing bacterial spot disease. 2. Using published computational pipelines, the conserved effector genes will be identified. 3. The conserved genes will be cloned and used as probes to identify new sources of resistant germplasm. 4. CRISPR-Cas9 genome editing strategies will be employed to make mutations in the orthologous bs5 gene and dmr6 genes in tomato.
Project Methods
For the genomic sequencing of the natural field isolates of Xanthomonas spp. isolated from infected tissue high through-put next generation seqeuncing will be performed by sequencing 96 strains that have been labelled with a unique bar code. The genomes will be assembled and effectors identified using the CLC software package. For the genome editing of the dmr6 and bs5 loci guide RNAs have been designed and will be cloned to Agrobacteriium binary vectors that contain the Cas9 protein under the control of the 355 promoter. Tomato plants will be transformed and mutants will be identified by sequencing the genes to determine the nature of the mutation.

Progress 10/01/16 to 09/30/21

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Undergraduates , graduate students and a postdocoral fellow were trained during this period. How have the results been disseminated to communities of interest?The information has been published in a peer-reviewed jopurnal. 2021. Daniela Paula de Toledo Thomazella, Kyungyong Seong, Rebecca Mackelspronag, Douglas Dahlbeck, Yu Geng, Upinder S. Gill, Tiangong Qi, Julie Pham, Priscilla Giuseppe, Clara Younga Lee, Arturo Ortega, Myeong-Je Cho, Samuel F. Hutton and Brian Staskawicz. Loss of fundtion of a DMR6 ortholog in tomato confers broad-spectrum disease resistance. PNAS July 6, 2021 118 (27) e2026152118; https://doi.org/10.1073/pnas.2026152118 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Significant progress was made in deploying CRISPR-Cas9 gene editing in tomato to make tomato plants resistant to Xanthomonas spp. We have shown that CRISPR-Cas9 induced mutations in either of the tomato bs5 gene or the tomato dmr6 gene give rise to resistant plants. Field trials were conducted with the tomato dmr6 plants demonstrating that these plants were significantly resistant to the bacterial spot disease.

Publications


    Progress 10/01/19 to 09/30/20

    Outputs
    Target Audience:The primary target of our research is other research scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we plan to solve the CryoEM strcuture of several NLR resistosomes to uncover the molecular basis of plant-pathogen speciticity. These data hopefully will allow us to design and generate novel NLR disease resistance genes that will be durable to invadinf pathogens.

    Impacts
    What was accomplished under these goals? Nucleotide?binding and leucine?rich repeat immune receptors (NLRs) provide resistance against diverse pathogens. To create comparative NLR resources, we conducted resistance gene enrichment sequencing (RenSeq) with single?molecule real?time sequencing of PacBio for 18 accessions in Solanaceae, including 15 accessions of five wild tomato species. We investigated the evolution of a class of NLRs, CNLs with extended N?terminal sequences previously named Solanaceae Domain. Through comparative genomic analysis, we revealed that the extended CNLs (exCNLs) anciently emerged in the most recent common ancestor between Asterids and Amaranthaceae, far predating the Solanaceae family. In tomatoes, the exCNLs display exceptional modes of evolution in a clade?specific manner. In the clade G3, exCNLs have substantially elongated their N?termini through tandem duplications of exon segments. In the clade G1, exCNLs have evolved through recent proliferation and sequence diversification. In the clade G6, an ancestral exCNL has lost its N?terminal domains in the course of evolution. Our study provides high?quality NLR gene models for close relatives of domesticated tomatoes that can serve as a useful resource for breeding and molecular engineering for disease resistance. Our findings regarding the exCNLs offer unique backgrounds and insights for future functional studies of the NLRs

    Publications

    • Type: Journal Articles Status: Published Year Published: 2020 Citation: 2020 Kyungyong Seong, Eunyoung Seo, Kamil Witek, Meng Li, and Brian Staskawicz. Evolution of NLR resistance genes with noncanonical N-terminal domains in wild tomato species. New Phytologist Volume 227, Issue 5 First published: 28 April 2020 2019. Shane Horsefield, Hayden Burdett, Xiaoxiao Zhang, Mohammad K. Manik, Yun Shi, Jian Chen2,3, Tiancong Qi, Jonathan Gilley, Jhih-Siang Lai, Maxwell X. Rank, Lachlan W. Casey, Weixi Gu, Daniel J. Ericsson, Gabriel Foley, Robert O. Hughes, Todd Bosanac, Mark von Itzstein, John P. Rathjen, Jeffrey D. Nanson1, Mikael Boden, Ian B. Dry, Simon J. Williams, Brian J. Staskawicz, Michael P. Coleman, Thomas Ve, Peter N. Dodds , Bostjan Kobe. NAD+ cleavage activity by animal and plant TIR domains in cell death pathways. Science: Vol. 365, Issue 6455, pp. 793-799


    Progress 10/01/18 to 09/30/19

    Outputs
    Target Audience:The primary target of our research is other research scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We have identified CRISPR-Cas9 induced mutants in the tomato bs5 gene. These plants will be characterized and analyzed for disease resistance. These plants will also be field tested for disease resistance to bacterial spot in Florida.

    Impacts
    What was accomplished under these goals? Genetic mapping and viral?induced gene silencing were used to identify immune signaling components of the XopJ4 perception pathway in N.benthamiana. Transient complementation assays were performed to determine the functionality of gene variants and co?immunoprecipitation assays were used to gain insight into the molecular mechanism of the pathway. Two N.benthamiana ethyl methanesulfonate (EMS) mutants deficient for XopJ4 perception were identified as having loss?of?function mutations in the gene encoding the nucleotide binding, leucine?rich repeat (NLR) protein NbZAR1. Silencing of a receptor?like cytoplasmic kinase family XII gene, subsequently named XOPJ4 IMMUNITY 2 (JIM2), blocks perception of XopJ4. This study demonstrates the feasibility of conducting mutant screens in N.benthamiana to investigate the genetic basis of the plant immune system and other processes. The identification of NbZAR1 and JIM2 as mediating XopJ4 perception in N.benthamiana supports the model of ZAR1 being involved in the perception of many different pathogen effector proteins with specificity dictated by associated receptor?like cytoplasmic kinases.

    Publications

    • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Alex Schultink, Tiancong Qi,Julia Bally, and Brian Staskawicz 2019 Using forward genetics in Nicotiana benthamiana to uncover the immune signaling pathway mediating recognition of the Xanthomonas perforans effector XopJ4. New Phytologist Volume221, Pages 1001-1009


    Progress 10/01/17 to 09/30/18

    Outputs
    Target Audience:The primary target of our ausience is other research scientist Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We plan to isolate and solce the structure of the Roq1/XopQ signaling complex be employing cryo-EM.

    Impacts
    What was accomplished under these goals? We performed CRISPR-Cas9 induced mutagenesis of the Nicotiana benthamiana NRG1. Mutations in this gene compromised the Roq1 disease reistance signaling pathway as well as other TNL immune receptor signaling pathways such as RPP1.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2018 Citation: NRG1 functions downstream of EDS1 to regulate TIR-NLR-mediated plant immunity in Nicotiana benthamiana. Tiancong Qi, Kyungyong Seong, Daniela P. T. Thomazella, Joonyoung Ryan Kim, Julie Pham, Eunyoung Seo, Myeong-Je Cho, Alex Schultink, and Brian J. Staskawicz PNAS 1814856115


    Progress 10/01/16 to 09/30/17

    Outputs
    Target Audience:The primary target of our audience is other research scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The results have been talked about in a meeting at Cold Spring Harbor about plant NLR genes this past September. What do you plan to do during the next reporting period to accomplish the goals?We plan to investigate the Roq1 signal transduction pathway and introduce the Roq1 gene in other plants to test for expression

    Impacts
    What was accomplished under these goals? We have employed the XopQ effector expressed in Xanthomonas spp. to find the corresponding Resistance gene Roq1 in Nicotiana benthamiana. The Roq1 gene has been introduced in tomato and show resistance to various strains of Xanthomonas species that infect tomato. We also plan to test genome edited tomatoes in both bs5 and dmr6 for resistance to bacterial spot disease.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2017 Citation: 2017. Schultink, Alex, Tuancing Qi, Arielle Lee, Adam Steinbrenner and Roq1 mediates recognition of the Xanthomonas and Pseudomonas effector proteins XopQ and HopQ. The Plant Journal10.1111/tpj.13715