Dissecting the defence responses activated by tomato receptors able to detect Fusarium oxysporum f. sp. lycopersici infection

Background

The soil-borne vascular-wilt fungus Fusarium oxysporum is one of the world’s most notorious fungal pathogens of crop plants because it is a species complex that combines the ability to cause severe yield losses with a wide host range overall, affecting diverse crops such as tomato, cotton, legumes, melons and banana (plantain). However, deployment of resistance genes can provide effective control of vascular-wilt diseases caused by soil-borne fungi. The interaction between tomato and Fusarium oxysporum f. sp. lycopersici (Fol) serves as a powerful model system in which to study the molecular basis of resistance and susceptibility in plants against vascular-wilt pathogens because of the recent identification of both host and pathogen genes controlling the interaction and the genomic resources available for both. Currently, four resistance genes have been introgressed into commercial tomato cultivars to protect against Fol infection and they have been named I (for Immunity), I-2, I-3 and I-7. These four genes have all been cloned. I and I-7 encode leucine-rich repeat receptor-like proteins (LRR-RLPs) and I-3 encodes an S-receptor-like kinase. I-2 encodes an intracellular receptor protein belonging to a major class of nucleotide-binding leucine-rich repeat (NB-LRR) resistance proteins that typically detect pathogen effectors delivered to the host cytoplasm either directly or via alterations to the cytoplasmic host proteins they target. It has been reported that EDS1 (Enhanced Disease Susceptibility 1) is required for I and I-7-mediated resistance, partially required for I-3-mediated resistance but not required at all for I-2-mediated resistance. Hence, it is crucial to identify the plant genes involved in signalling and delivery of tomato receptor-mediated resistance, which will increase our understanding of the molecular mechanisms underlying Fol resistance.

Aims

The overall goal of this project is to discover how the tomato plant delivers effective resistance against Fol infection. To answer this question, we want to:

1) identify specific genes involved in resistance delivered by membrane-localized receptors including I, I-3 and I-7, respectively;

2) identify specific genes involved in delivering cytoplasmic NB-LRR receptor I-2-mediated resistance;

3) employ CRISPR/Cas9 genome-editing to generate tomato plants carrying deletions of candidate genes identified in Aims 1 and 2;

4) test the roles of candidate genes in tomato/Fol interaction by disease assay.

Methodology and Work Plan

Comparative RNA-sequencing to identify differentially expressed genes in response to Fol

This project will use deep RNA-sequencing to identify the subsets of tomato genes induced specifically in the context of I-, I-2-, I-3- and I-7-mediated resistance, respectively. Ten-day-old seedlings of the tomato cultivar Moneymaker (MM), which lacks any genes for Fol resistance, and a near-isogenic cultivar carrying only I or I-2 or I-3 or I-7 resistance gene will be inoculated with Fol carrying its corresponding Avr gene. Mock-inoculated plants will be included as controls. RNA will be extracted from the roots of ten plants per treatment at 2, 4 and 8 days after inoculation. This experiment will be repeated three times to generate biological replicates. Transcript profiles of the infected and mock-inoculated roots will be determined by RNA-seq analysis using the Illumina HiSeq 2000 platform. Data analysis will be performed using the CLC Genomics Workbench and the Bioconductor package DESeq2 v. 1.6.3. Differential gene expression analysis will be performed by comparing Fol- and mock-inoculated plants for each cultivar to filter out reads associated with the inoculation process, and then by comparing the filtered transcripts between MM and the near-isogenic cultivars each carrying a single Fol-resistance gene.

Label-free quantitative-proteomics to identify differentially expressed gene products

As a complementary approach to the RNA-seq analysis, MaxQuant label-free quantitative-proteomics mass-spectroscopy will be used to obtain independent confirmation of the genes induced by tomato receptor-mediated resistance. This method has been used successfully to analyse the xylem proteome in susceptible tomato following infection by strains of Fol carrying individual effector gene deletions. However, this work did not address the resistance response and only identified proteins represented in the xylem sap, whereas plant responses would also be expected to include cell surface and intracellular plant proteins. Total protein will be isolated and analysed from both resistant and susceptible tomato challenged with Fol. The experimental setup and sample collection times will be the same as those used for the RNA-seq experiment. Total protein isolated from each root sample will be subject to in-gel/solution tryptic digestion and the tryptic peptides analysed by Thermo LTQ–nanoLC-MS/MS mass spectrometry. The raw data from the LTQ-Orbitrap will be analysed with MaxQuant software to identify proteins for label-free relative quantification. The tomato protein reference database will be obtained from the tomato SOL genomics network website (https://solgenomics.net/). Bioinformatic analysis of the MaxQuant workflow and statistical analysis of protein abundance will be performed using Perseus software available at the MaxQuant website (http://www.coxdocs.org/).

Employing CRISPR/Cas9 to knockout candidates genes identified and pathology test

In recent years, the CRISPR/Cas9 technique has become a feasible and practical approach for generating gene deletions that can be used to validate gene function. CRISPR/Cas9 has several advantages over conventional RNA interference-mediated gene-silencing methods such as hairpin RNA-mediated silencing or virus-induced gene silencing, which have various associated problems such as variable knockdown efficiency and off-target effects. To generate CRISPR/Cas9 gene-deletion constructs, this project will take advantage of available vectors that have already been used successfully in tomato. The standard Agrobacterium-mediated cotyledon transformation method will then be used to generate transgenic tomato plants carrying CRISPR/Cas9 constructs targeted against genes identified in Aim 1 and 2. Gene deletions will be confirmed by PCR amplification and sequencing, and progeny of homozygous deletion lines will be inoculated with Fol to determine whether the deletions have any effect on receptor-mediated resistance.

Supervisors:   Assoc. Prof. David Jones and Dr Lisong Ma