Performing Department
Graduate Studies
Non Technical Summary
Lettuce is a major vegetable crop in the United States, with a total production value of $2.9 billion in 2016. Production of lettuce is threatened by susceptibility to various pathogens, the most important of which is lettuce downy mildew caused by the oomycete, Bremia lactucae. Downy mildew thrives in areas with cool temperatures and high humidity, such as the California coastal valleys where the majority of US lettuce is grown. Infection can lead to death of seedlings and can cause necrotic lesions, secondary infections, and loss of marketable yield in older plants. The most effective control of lettuce downy mildew is obtained by planting of resistant cultivars. Current lettuce breeding programs are constantly breeding for new varieties of disease resistant lettuce, primarily by using introgression from wild lettuce accessions to bring in novel downy mildew resistance genes. In recent years, knowledge of the genetic and molecular basis of plant-pathogen interactions has accelerated plant breeding for disease resistance. Effectors, which are plant pathogen-derived proteins secreted into plant cells to alter host processes, can be recognized by plant receptors encoded by resistance genes, triggering an immune response and resulting in host resistance. This research project aims to use effectors from lettuce downy mildew as molecular probes to uncover the genetic basis of disease resistance in lettuce. . In addition to discovering genes underlying downy mildew resistance in lettuce, these results will inform what genes are important in the pathogen for determining compatibility, which will enable pathogen monitoring and more informed deployment of resistance genes for improved plant health and agricultural production of lettuce.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
The major goals of this project are to (1) identify effectors in lettuce downy mildew (Bremia lactucae) that are recognized by resistant and/or wild lettuce accessions and (2) to use downy mildew effectors as tools to map and characterize lettuce downy mildew resistance genes. Knowledge of effectors can then be used in population monitoring of lettuce downy mildew in the field and identification of novel resistance genes can be used in breeding and biotechnology to create new resistant varieties of lettuce.
Project Methods
i)_Comparative genomics to identify candidate effectors: The candidate B. lactucae effectors are predicted on the basis of presence of the WY domain, a structural domain identified in Phytophthora species shown to contribute to virulence and avirulence activity of effectors. I used a Hidden Markov Model build on sequences from three Phytophthora species to search for WY domain containing proteins in B. lactucae. My candidate effectors show other features of effectors, such as N-terminal secretion signals, lineage specificity to Bremia, and expression during infection. ii) Candidate effector cloning: I have previously used Gateway cloning to amplify seven candidate effectors from one isolate of B. lactucae (SF5). During this process, I optimized each step of the cloning workflow and I am now poised to do high-throughput Gateway cloning on the remaining 48 effector candidates. I plan on cloning candidates from two isolates of B. lactucae: one European isolate and one Californian isolate. The two isolates have different virulence profiles and therefore are expected to contain sequence divergence in recognized effectors. iii)_Germplasm Screening: Effector candidates will be cloned into the Gateway compatible pEG100 vector for plant expression. Expression vectors will be transformed into Agrobacterium tumefaciens strain C58 for agroinfiltration and transient expression in lettuce. A diverse germplasm panel of 64 cultivated and wild lettuce varieties representing all known Dm genes will be screened for effector recognition by agroinfiltration. Briefly, the youngest fully expanded leaves of 3 to 4-week old lettuce plants will be infiltrated with Agrobacterium using a needless syringe. Leaves will be examined 4-5 days post infiltration for signs of macroscopic cell death, indicative of immune recognition by host resistance proteins. In my experience, this assay can be prone to false positives, therefore effector recognition will be confirmed by repeating infiltrations on at least six plants for lines that are found to recognize effectors. In addition, as a negative controls, I will use empty vector and pEG100:GFP containing Agro. For positive controls, I will use HopM1 and AvrPto, Pseudomonas effectors and known elicitors of cell death in lettuce. Using this screening method on seven candidate effectors, I previously identified two related lettuce varieties with introgressions from L. virosa, ViAE and ViCQ, that recognized three B. lactucae effectors: SW1, SW3, and SW4. Another variety of lettuce, Ninja, was found to also recognize SW4 (unpublished data). Based on the success of this initial screening experiment (3/7 effectors recognized), I expect to find many instances of effector recognition in my screen of the additional 48 effector candidates. The genetic basis of any newly discovered effector recognition will be characterized by generating F2 mapping populations and performing genotyping-by-sequencing on segregating progeny.iv)_Effector recognition mapping: I have previously used an F2 mapping population of Ninja x Valmaine to map recognition of SW4 in Ninja to a 4 Mb region on Chromosome 4 (unpublished data). I will do similar experiments for any new lettuce varieties that are found to recognize effectors. If one is not already available, I will cross these varieties with the susceptible variety Cobham Green (CG) to generate an F2 mapping population segregating for the effector recognition phenotype. I will score 192 F2 progeny for effector recognition and collect leaf tissue DNA extraction for GBS. I will use high-throughput protocols optimized for lettuce for DNA extraction and library prep that are routinely used in the Michelmore lab. Low coverage sequencing will be performed on the Illumina HiSeq 4000 with 100 bp paired-end reads. Reads will be mapped to the lettuce reference genome to generate high quality SNP markers. Markers will be clustered based on the lettuce genetic map and linkage maps will be developed using MSTMap.For mapping the recognition of SW1, SW3, and SW4 in ViAE and ViCQ, a different approach will be used. There is already a recombinant inbred line (RIL) population that was generated from ViAE x CG and ViCQ x CG to map downy mildew resistance, which was found to map to chromosome 8 (Maisonneuve, B and Truco, M, unpublished data). I will perform phenotyping and GBS on this RIL population to map effector recognition and to determine if any of the loci associated with effector recognition overlap with the previously identified locus underlying downy mildew resistance.v)_Resistance gene family identification: The Michelmore lab has generated a collection of lettuce RNAi tester lines that contain silencing constructs against all of the major NLR families in lettuce. I will cross Ninja, ViAE, and ViCQ to RNAi tester lines that target NLRs within the recognition locus. I will phenotype F1's from the RNAi crosses by infiltrating with the recognized effector. If recognition is lost, then it implicates the silenced NLR family in recognition of that effector. I will test for efficiency of silencing using transient GUS assays, since all RNAi tester lines also have a trigger sequence for GUS silencing.vi)_Identification of causal genes by knockout and complementation: Once NLR families have been identified for Ninja and ViAE/ViCQ, I will use CRISPR/Cas9 mediated knockout of the candidate genes. I will use Cas9 and sgRNA delivery vectors (Bertier et al, manuscript in prep) that have shown to be effective in lettuce in the Michelmore lab for Agrobacterium-mediated transformation of Ninja or ViAE at the Ralph M. Parsons transformation facility at UC Davis. While the transgenic lettuce lines are being generated (4-6 months), I will clone candidate genes into the pEG100 expression vector for transient expression using gene synthesis of intronless candidate genes and Gateway cloning. Knockout events will be confirmed by amplicon sequencing of the gene of interest and non-functional mutants will be used for phenotyping by effector recognition. Loss of effector recognition due to knockout would implicate the gene in recognition, which will then be confirmed by complementation with the functional gene in a transient assay.Evaluation Plan: Due to the large number of effectors and varieties being tested in the germplasm screen, the screen will take place in batches over the course of four months. Completion of the germplasm screen will be a major milestone for the project. Completion of mapping experiments and identification of NLR families from the RNAi crosses will be subsequent major milestones. Mentoring milestones will primarily be assessed by presentations at meetings and by manuscript preparation.Efforts: The Michelmore lab has close connections to seed companies and lettuce growers across California and internationally. Advanced breeding lines with novel resistance genes will be shared with these stakeholders. Knowledge of downy mildew effectors will be shared through the Michelmore lab website "Bremia database" which is already used by growers to know which pathotypes are prevalent in a given area and year. Knowledge of lettuce downy mildew resistance genes will also be shared with stakeholders at the annual California Leafy Greens Research Board meeting.