Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Plant Pathology
Non Technical Summary
Black rot of crucifers, caused by the bacterium Xanthomonas campestris pv. campestris (Xcc), is one of the most important diseases of cabbage in the US and worldwide.Control measures for black rot are limited and include copper application, crop rotation, removal of crop debris and cruciferous weeds, and using pathogen free seed.While there is variation in crucifer tolerance to black rot, there is limited understanding of the mechanisms of resistance or susceptibility. To find sources of resistance, it is vital to gain a better understanding of Xcc virulence factors and what they are targeting in the plant host. Identification of genes that lead toBrassicasusceptibility could inform targeted breeding strategies to grow more disease resistant crops.A critical component to Xcc pathogenicity is the type III secretion system (T3S) which is a syringe like structure thatinjects bacterial "effector"proteins into the plant host cell. Once inside the cell, these effectors target host protein and nucleic acids in the plant to improve conditions for bacterial growth. A large family of T3S effector proteins, the transcription activator-like effectors (TALEs), are found in many Xanthomonas species and in other Xanthomonas/crop systemsare known to target genes in the plant called Susceptibility (S) genes to help the bacterium survive.Due their recent discovery in Xcc strains, the contribution of TALEs to Xcc virulence is unknown.In this project we propose to determine the presence of TALEs in a collection of Xcc isolates, identify what S genes they are targeting in the brassica host, and whether they are contributing to bacterial virulence. Finally, we will investigate whether changing these genes, so that Xcc can no longer use them, will result in a more black rot resistant cabbage.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
This project has goals of research progress and sceintific training in effector biology and host susceptibility for Zoe Dubrow, a PhD candidate in plant pathology at Cornell University.Black rot caused by Xanthomonas campestris pv. campestris is one of the most important brassica diseases worldwide. While there is variation in brassicatolerance to black rot, there is limited understanding of the mechanisms of resistance or susceptibility. A critical component to Xcc pathogenicity is the type III secretion system (T3S) which translocates bacterial effector proteins into the plant host cell. Once inside the cell, these effectors target host protein and nucleic acids in the plant to improve conditions for bacterial proliferation. A large family of T3S effector proteins, the transcription activator-like effectors (TALEs), localize to the nucleus of the host cell to directly bind specific plant gene promoters and upregulate these genes via a C-terminal activation domain. Due their recent discovery in Xcc strains, the contribution of TALEs to Xcc virulence is unknown. The major goal of this project is to find sources of brassica susceptibility to Xcc, by identifying and characterizing TALE targeted susceptibility genes.Research Objectives Determine the distribution and diversity of TALEs from NY State Xcc isolates Quantify the virulence contributions of Xcc TALEsIdentify TALE targeted susceptibility genesMeasure broad-spectrum dependence on basal susceptibility gene expressionGraduate Training Objectives1. Attend at two conferences to present research outlined in this proposal2. Publish 2manuscripts on the proposed research3. Completion of the graduate degreeat the end ofthe project duration
Project Methods
Methods:Determine distribution and diversity of TALEs from NY State Xcc isolatesMethods: We have determined TALE presence in all New York isolates of Xcc in the Smart lab collection by PCR, Southern, and western blotting. Next, we will begin to sequence Xcc TALEs from all TALE containing isolates using replicated sanger sequencing of PCR amplicons. Genome sequences for selected isolates, chosen based on results of Southern and western blots, will be produced using Pacific Biosciences SMRT sequencing, to generate long reads to resolve TALE genes. Following sequencing, we will determine the genomic context of each TALE to see whether they are plasmid borne or chromosomal, or surrounded by IS elements. We will then use the computational tool FuncTAL to predict whether TALEs have similar target groups[21].Expected Outcomes: From this objective, we will determine the TALE content of a large collection of Xcc isolates from NY. Preliminary sequencing data has shown that certain TALEs are conserved among multiple isolates. This is especially likely between closely related isolates, but may indicate that a particular TALE is important if it is conserved across many less similar isolates. Based on the uneven distribution of TALEs, it is expected that many will be plasmid borne or surrounded by IS elements.Contingency Plans: PCR amplification of genes, particularly repetitive genes like TALEs, can introduce errors in sequences. Therefore, each TALE gene will be amplified and sequenced at least twice. Strains with large TALEs that cannot be Sanger sequenced in their entirety will be selected for whole genome sequencing.Quantify the virulence contributions of Xcc TALEsMethods: To determine whether individual TALEs increase Xcc virulence, we are using a deletion strategy described in [22]. We have successfully knocked out TALEs from 6 isolates and will create at least 10 more knock out strains for TALEs with disparate predicted binding specificities. Isolates are chosen for mutagenesis based on their TALE content with a preference towards isolates expressing a desired TALE and lacking other TALEs. By choosing isolates with less TALEs, we hope to avoid issues of redundancy in TALE function that could mask virulence phenotypes. We will conduct clip infiltration assays using wildtype and TALE deletion strains on two commercially relevant varieties of cabbage (Surprise and Capture), cauliflower (Skywalker), and Turnip (Seventop). These crops were chosen because cabbage is the most economically important cole vegetable in NY, cauliflower is the most susceptible, and turnip is closely related Brassica species (Brassica rapa) that is also susceptible to black rot. Symptoms will be recorded quantitatively by measuring lesion length using time points described in [23]. For strains where the mutagenized version has a different virulence phenotype to wildtype, mutagenized strains will be complemented by transforming a standard expression plasmid containing the cloned TALE. Virulence assays will be repeated comparing the deletion, complemented, and wildtype strains.Expected Outcomes: When TALEs are important to virulence, we expect plants infected with TALE deletion strains to have reduced symptoms and bacterial growth, corresponding to the contribution of the TALE, compared to those infected with the wildtype strain. Complementation of the TALE should restore wildtype symptoms and growth. Differential virulence may only be apparent on certain hosts based on the presence of EBEs.Contingency plans: It is possible that none of the TALEs tested in this experiment will have a measurable effect on virulence. In this case, we will expand our mutagenesis screen to include 10 more TALEs. We may also expand virulence assays to other Brassica hosts or test other inoculation methods to determine if TALEs are involved in host range or colonization of specific tissues. If none of the TALEs tested are virulence determinants, for objective 3 we will focus instead on hax2, hax3, and hax4 which are Xcc TALEs known to increase virulence in multiple hosts[18].Identify Xcc TALE targeted susceptibility genes Methods: We will begin to identify S genes targeted by TALEs through an RNA-seq experiment completed by the PD at LIPM in Toulouse, France in the lab of Dr. Laurent Noel. Cloned TALEs of interest will be transformed into a reference strain of Xcc (e.g. Xcc 8004) that lacks TALEs. The wildtype and TALE expressing strains will be inoculated on susceptible cauliflower and cabbage. Tissue will be sampled 48 hours post inoculation and RNA will be extracted, prepared, and submitted for RNA-seq. In some cases, we may be able to leverage RNA-seq data already produced by the Noel lab, where these data have already been collected for multiple Xcc TALEs from global collections. TALE sequences will be input into TALE-NT 2.0 to predict binding sites in cabbage promoter regions[24]. We will compare genes upregulated in plants infected with the TALE expressing strains from the RNA-seq experiment with the computationally predicted targets. Genes that appear in both data sets will be evaluated based on function and level of upregulation to determine likely targets. These targets will be validated via qRT-PCR using primers specific to the target. Two designer TALEs (dTALEs), DNA sequences constructed in the lab to mimic TALE effectors, but with alternate RVD sequences, per target gene will be designed to individually induce expression of suspected S genes. These dTALEs will be transformed into the strain of origin of the TALE and used to infect susceptible Brassica.Expected Outcomes: We expect to find likely TALE targets in the differentially regulated genes in the RNA-seq experiment that also appear in the TALE-NT 2.0 predicted target list, as we have in past pathosystems. For each TALE that increases virulence, we expect to find at least one corresponding S gene target. Multiple TALEs with different RVD sequences may target a single gene promoter. Likely targets include SWEETs, transcription factors, and genes from other previously identified S gene families. However, S genes identified in this study may be unique from TALE targets observed in other pathosystems. The expression of dTALEs targeting S genes are expected to restore TALE deletion strains to full virulence.Contingency Plans: The efficiency of dTALEs upregulation of a target is often different than the native TALE, which is why we will use at least two. If S gene function is dose dependent, then this could be an issue. If needed, we will design additional dTALEs to more closely mimic native conditions.Measure broad-spectrum dependence on basal susceptibility gene expressionMethods: We will use virus induced gene silencing (VIGS) to silence S genes discovered in aim 3 in susceptible cabbage [25, 26]. Silenced and control plants will be infiltrated with 10 diverse Xcc isolates. Lesion length will be measured as described in aim 2. S gene expression will be measured via qRT-PCR.Expected Outcomes: If basal S gene expression is broadly important to Xcc, then it is expected that most or all Xcc isolates infiltrated onto S gene silenced plants will multiply less and cause fewer symptoms. This outcome may be gene dependent and may be true for some S genes and not others.Contingency plans: If silencing of S genes is unsuccessful in cabbage, these assays will instead be conducted on Arabidopsis thaliana, which is in the Brassicaceae family.