Progress 05/01/24 to 04/30/25
Outputs
Target Audience:US plant pathology community belonging to American Phytopathological Society Southern Division; Florida secondary school teachers; Faculty, staff and students in Department of Plant Pathology, University of Florida;Faculty, staff and students in Department of Plant Pathology, University of Georgia; Plant science community at University of Georgia. Changes/Problems:Due to the relocation of PI Tian's lab andthe transfer of the award from University of Hawaii to University of Georgia, the execution of the project was slightly delayed. We were not able to provide the summer internship to a high school student during the summer of 2024, but we will provide the internship opportunities to two high shool students during the summer of 2025. What opportunities for training and professional development has the project provided?This project has provided research opportunities mainly to three PhD students with one at University of Florida (UF) and two at University of Georgia (UGA). They gained skills in plant pathology and molecular biology. PhD student Alexander Fast at UF obtained a 3-year FFAR fellowship for additional professional development. Two additional graduate students from University of Florida received training in Agrobacterium-mediated transformation in the Tian lab and graduate student Rana Elessawy is implementing the protocol in the Goss lab. The project alsoprovided hands-on research experiences to three undergraduate students, include an undergraduate student at UGA for a summer research internship in 2024, one from Abraham Baldwin Agricultural College, and one from UF. In addition, the project provided training opportunities to secondary school teachers by hosting a one-day "Plants get sick too!" workshop for 18 Florida middle and high school teachers in May 2024, at which they were introduced to our research onPhytophthora. How have the results been disseminated to communities of interest?PhD student Alexander Fast from University of Florida presented at the Joint Meeting of the Caribbean andSouthern Division of the American Phytopathological Society, March 9-13, 2025. PI Tian presented two invited talks, with one to the department of Plant Pathology at University of Florida and another at Plant center retreat organized by University of Georgia (UGA). One PhD student at UGA presented a poster at annual meeting of the Georgia Association of Plant Pathologists (GAPP), March 4-5, 2025. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. Determine the nuclear-localized molecular interaction dynamics of PpalRxLR1 and the subverted host cellular processes during infection. To complete this objective, aPhytophthora palmivorastrain with fluorescently tagged PpalRxLR1 is essential. To generateP. palmivorastrains with endogenous PpalRxLR1 tagged with GFP through homology-directed repair (HDR) using CRISPR-Cas9 reagents, a 20-nt sgRNA target sequence with the Cas9 cleavage site close to the stop codon of PpalRxLR1 was selected, and cloned to pYF515, an "all-in one" plasmid containing both Cas9 and sgRNA expression cassettes for oomycete gene editing. The donor DNA template for inserting the enhanced GFP (EGFP) coding sequence to the immediate upstream of PpalRxLR1 stop codon, which includes 1kb of gDNA sequence upstream of the stop codon, EGFP coding sequence, and 1kb of gDNA sequence containing the stop codon and downstream sequence, was produced using Gibson Assembly of three PCR fragments, and cloned to pJET1.2/blunt in ConeJET PCR Cloning Kit. We co-transformedP. palmivoracacao isolate HI1781 with the plasmid expressing Cas9 and sgRNA and the plasmid harboring the donor DNA template via electroporation of zoospores. We were able to obtain transformants, but none of them had PpalRxLR1 tagged with EGFP. As an alternative approach, we have generated P. palmivora strains ectopically expressing PpalRxLR1-EGFP. To avoid the silencing of the introduced PpalRxLR1-EGFP by the endogenous copy of PpalRxLR1 and its homologs, we synthesized a codon-modified PpalRxLR1 (cmPpalRxLR1) which does not share significant similarity with the wild-type PpalRxLR1 at the nucleotide sequence level. These strains are currently being evaluated for their infectivity, growth and development. A suitable strain will be selected and used to determine the subcellular localization of PpalRxLR1 during plant infection by confocal microscopy and its host targets through ChIP-seq and Co-immunoprecipitation. To determine the host processes subverted by PpalRxLR1, we planned to perform comparative transcriptomic analyses using bothNicotiana benthamianaand cacao. ForN. benthamiana, we transiently expressed DsRed and DsRed-PpalRxLR1 in leaves viaAgrobacterium-mediated transient expression. The expression of DeRed and DsRed-PpalRxLR1 was confirmed using confocal fluorescence microscopy, which showed the localization of DsRed-PpalRxLR1 in the nucleus and DsRed in both cytoplasm and nucleus. mRNA sequencing was performed with samples expressing either DsRed or DsRed-PpalRxLR1, with each treatment having three biological replicates. A total of 602 differentially expressed genes (DEG) were identified, with 341 downregulated by PpalRxLR1 and 261 upregulated. KEGG enrichment analysis found that the most highly enriched pathways included MAPK signaling pathway, plant-pathogen interaction and phenylpropanoid biosynthesis, suggesting that PpalRxLR1 contributes to pathogenicity by modulating plant defense. For analyses using cacao, we are in the process of optimizing the protocols for high level of transient gene expression. 2. Determine the role of PpalRxLR1 as a key component in host range evolution of P. palmivora. We assembled a diverse collection of isolates representing the geographic and host distribution of P. palmivora, including over 400 isolates from more than 30 plant families. We are currently confirming the identification of a subset of isolates that we will use for subsequent sequencing and experiments. Some isolates previously identified as P. palmivora are instead the closely related, recently described species Phytophthora heterospora. Understanding the host and geographic distribution of these species and their evolutionary relationship will clarify the population structure of P. palmivora. 3. Identify effectors with key roles in pathogenicity of P. palmivora on a broad range of hosts. We have been working on one RxLR effector, designated as PpalRxLR5. It is conserved in all 24 isolates with sequencing data available, abundantly expressed and highly induced during infection of papaya by aP. palmivorapapaya isolate and during infection of cacao by a cacao isolate, as determined by RT-qPCR. To determine its role in pathogenicity using mutants generated via CRISR/Cas9 genome editing, a single guide RNA (sgRNA) was designed and cloned to pCB301TOR-CRISPR, a vector used for oomycete genome editing viaAgrobacterium-mediated transformation. The resulted plasmid was used to transform aP. palmivorapapaya isolate. Over 60 G418-resistant transformants were recovered. From 25 single zoospore transformants that were produced from 12 original transformants, three mutants were identified. Infection assays with these mutants and the wild-type strain are currently on-going. In addition, we used dual RNA-seq data from cacao pods experimentally infected with P. palmivora zoospores to identify additional genes upregulated during plant infection as candidate genes to explore for their roles in pathogenicity.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Fast AC, Rosano A, Tian M, Goss E. (2025) Multi-locus sequence typing of a diverse collection of Phytophthora palmivora isolates and the newly described Phytophthora heterospora Insights into global distribution. The Joint Meeting of the Southern and Caribbean Divisions of the American Phytopathological Society, March 9-13, 2025, Gainesville, FL.
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