PATHOGENICITY MECHANISMS AND CONTROL OF ECONOMICALLY IMPORTANT PLANT PATHOGENIC OOMYCETES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1020611
• Project Start Date: Oct 1, 2019
• Project End Date: Sep 30, 2024
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822

Performing Department
Plant & Environmental Protection Sciences

Non Technical Summary
Basil downy mildew and Phytophthora blight of papaya, caused by oomycete pathogens Peronospora belbahrii and Phytophthora palmivora respectively, are the major constraints severely affecting Hawaii's basil and papaya production. The overall goal of this project is to develop their control measures, and meanwhile dissect the molecular mechanisms of disease development in order to develop targeted genetic and chemical control in the future. The most effective strategy to control basil downy mildew is to use disease resistant sweet basil varieties, which are currently in shortage. To develop downy mildew resistant sweet basil, we have generated transgenic lines targeting various candidate genes potentially involved in disease development and plant resistance. These lines will be evaluated for downy mildew resistance in the lab and field trials and subjected to molecular analyses to identify highly resistant plants and understand the molecular basis of basil-P. belbahrii interactions. Fungicide application is by far the major control measure for Phytophthora blight of papaya, but suffers from lack of sufficient number of anti-oomycete fungicide alternatives for rotated use to avoid selection of fungicide resistant strains. Multiple natural metabolites shown to exhibit high inhibitory activities against P. palmivora during in vitro growth will be evaluated for their efficacy in controlling P. palmivora on plants. In order to understand P. palmivora pathogenicity mechanisms, a number of RxLR effectors will be investigated for their roles in pathogenesis, and detailed characterization of the effectors and their host targets will be performed for the ones that play a key role in pathogenicity.

Animal Health Component

40%

Research Effort Categories

Basic

60%

Applied

40%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	4099	1160	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1160 - Pathology;

Keywords

oomycetes

peronospora belbahrii

phytophthora palmivora

basil downy mildew

papaya

Goals / Objectives
This project will focus on two plant pathogenic oomycetes that threaten agricultural production in Hawaii and worldwide.The overall goal is to develop their control measures, and meanwhile dissect the molecular mechanisms of disease development in order to develop targeted genetic and chemical control in the future. The three specific objectives are: 1)Evaluate downy mildew resistance of sweet basil generated using biotechnology and understand the molecular basis of basil-Peronospora belbahrii interactions; 2)Evaluate the efficacy of newly identified anti-oomycete natural metabolites in control of Phytophthora blight of papaya; 3)Identify Phytophthora palmivora effectors that play key roles in pathogenesis and the underlying mechanisms.

Project Methods
Objective 1. Evaluate downy mildew resistance of sweet basil generated using biotechnology and understand the molecular basis of basil-Peronospora belbahrii interactionsWe have used three strategies to generate basil downy mildew resistance, including 1) overexpression of a basil putative resistance-related gene Oblectin1; 2) host-induced gene silencing (HIGS) of two P. belbahrii candidate RxLR effector genes PbEC1 and PbEC2, and PbORCE1 which encodes a putative endoplasmic reticulum (ER) resident protein that is highly conserved in all plant pathogenic oomycetes with genomic sequences available; and 3) CRISPR/Cas9-mediated gene editing of two basil candidate susceptibility (S) genes ObDMR1 and ObDMR6, the basil homologs of Arabidopsis DMR1 and DMR6, mutation of which conferred nearly complete resistance to Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis (van Damme et al., 2008; van Damme et al., 2009)We have generated T0 transgenic lines using Agrobacterium-mediated transformation for all targeted genes. For overexpression of Oblectin1, HIGS of PbEC1, PbEC2 and PbORCE1, we identified five T0 lines with relatively high level of transgene expression for each construct, and further identified the homozygous plants in T1 segregating populations derived from each selected T0 line by determining the antibiotics resistance and transgene copy numbers using quantitative PCR (qPCR). The homozygous T1 plants are growing for T2 homozygous seeds. For CRISPR/Cas9-mediated gene editing of ObDMR1, we have identified T1 plants from at least 3 initial T0 transgenic plants with all copies of ObDMR1 mutated and these plants are growing for T2 seeds. For gene editing of ObDMR6, we have generated several dozens of T0 transgenic plants, we will analyze T0 and T1 plants to identify the plants with all copies of ObDMR6 mutated, which will be propagated to get seeds for further analyses.Evaluation of downy mildew resistance of the homozygous transgenic plants or gene-edited plants described above will be performed initially in the lab conditions by spraying inoculation of sporangium suspensions of P. belbahrii. Leaf samples will be collected from a set of infected plants at 4 days post inoculation. Depending on the constructs, these samples will be used to determine the expression of transgenes and/or the level of silencing of targeted pathogen genes using reverse transcription quantitative PCR (RT-qPCR), and quantify the pathogen biomass using qPCR as described previously (Shao and Tian, 2018). Another set of infected plants will be kept for visual observation of disease symptoms. These results will allow us to determine the disease resistance levels of all transgenic plants and the roles of these selected P. belbahrii and basil genes in disease resistance and susceptibility.The plants with high level of downy mildew resistance based on lab tests will be subjected to field trials to evaluate their downy mildew resistance. Field trials will be performed in both Waimanalo Research Station and Poamoho Research Station of University of Hawaii at Manoa, where natural P. belbahrii inocula are ubiquitous. Disease resistance will be scored as described previously (Wyenandt et al., 2010). In addition, other phenotypic changes compared with wild-type will be recorded.Objective 2. Evaluate the efficacy of newly identified anti-oomycete natural metabolites in control of Phytophthora blight of papayaWe have identified five plant-derived natural metabolites with a low risk to the environment and human health that highly inhibit zoospore germination and mycelium growth of P. palmivora during initial screening and further in vitro growth assays. Their efficiency in controlling P. palmivora on plants will be tested to determine the potential to develop them into commercial fungicides. As infection of fruit and roots of young plants account for the most parts of economical losses caused by this disease, we will test the preventative and curative effects of these compounds on fruit and root infection by P. palmivora.For preventative effects on fruit infection, the solution of the compound in the presence of surfactant will be sprayed on fruit until running down; 1 day later, the treated fruit will be inoculated with P. palmivora by spraying zoospore suspensions. For curative effects on fruit infection, the fruit will be inoculated first and 1 day later the compound solution will be sprayed. A treatment with surfactant water solution will serve as a control. The development of disease symptoms will be monitored.For preventative effects on root infection, 6-week-old seedlings in pots will be drenched with the compound solution, 1 day later, the plants will be inoculated with P. palmivora zoospore suspension by depositing the zoospore suspension in a hole made in the soil 1-cm away from the main stem. For curative effects on root infection, the seedlings will be inoculated with P. palmivora zoosore suspension as described above, and then the compound solution will be drenched at 1-d, 2-d and 3-d post inoculation. For test of both preventative and curative effects, drenching with water serves as a negative control. The percentage of the plants that develop disease symptoms and the level of the disease will be recorded.Initial testing will be performed using a relatively high concentration. If the compounds prove effective, we will further test the efficiency using a series of concentrations and compare with a commercial anti-oomycete fungicide.Objective 3. Identify P. palmivora effectors that play key roles in pathogenesis and the underlying mechanismsFive to eight candidate RxLR effector genes shown to be most abundantly expressed and most highly induced during infection compared with in vitro mycelium growth will be selected based on published mRNA-seq data (Ali et al., 2017). Their expression during infection of papaya and cacao will be further tested by RT-qPCR. The ones whose induced expression during infection was confirmed will be subjected to further analyses. To test their roles in pathogenicity, we will generate mutants using CRISPR/Cas9-mediated gene editing via Agrobacterium-mediated transformation, a P. palmivora reverse genetics toolkit PI Tian's lab has established recently (Wu et al., 2016; Gumtow et al., 2018). Single zoospore transformants will be isolated from initial G418-resistant transformants. The mutations of the target gene will be detected by PCR amplification followed by sequencing. The single zoospore transformants with the targeted gene mutated will be used to infect papaya and cacao leaves and/or fruit together with the wild-type strain to determine the roles of the target gene in pathogenicity. For the genes that play a critical role in P. palmivora pathogenicity, we will identify its host targets using a yeast two-hybrid screen. The interactions of the effector with the candidate host targets will be confirmed by expressing the potential interacting partners fused with different tags in Nicotiana benthamiana via Agrobacterium-mediated transient expression followed by co-immunoprecipitation as described elsewhere (He et al., 2018). The identification of the host targets will reveal the host factors that are involved in disease development and plant resistance. In addition, the effector and the host target's biochemical function and host cellular localization will be further determined. The resulted information will serve as foundation for designing targeted genetic and chemical control strategies.

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

Outputs
Target Audience:Plant scientists working on both basic and translational research, extension plant pathologists, andagricultural industries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided hands-on trainings and professional development to three undergraduate students and two graduate students. How have the results been disseminated to communities of interest?The results have been disseminated to communities of interest through conference presentations, peer-reviewed publications and a book chapter. What do you plan to do during the next reporting period to accomplish the goals?For Objective 1, we willfurther evaluate the disease resistance of ObDMR1/ObDMR6 mutants, and transgenic T2 homozygous plants overexpressing Oblectin1 with optimized infection assay conditions in the lab, and meanwhile applying for permits for field trials. We will also perform molecular and biochemical characterization of Oblectin1 if its overexpression enhances basil downy mildew resistance. For Objective 2, we willevaluate the efficacy of two newly identified anti-oomycete natural metabolites in control ofPhytophthorablight of papaya For Objective 3, we will continue to generate mutants of PpalRxLR1 using CRISPR/Cas9 gene editing, followed by determining its role in pathogenicity.

Impacts
What was accomplished under these goals? Oomycetes include many destructive plant pathogens that threaten agriculture and natural ecosystems. Among them, basil downy mildew pathogenPeronospora belbahriiis a major constraint affecting basil production in US and worldwide, andPhytophthora palmivoracauses devastating diseases on many important crops in the tropics and subtropics. As currently available disease control methods are insufficient, we have been developing disease resistant sweet basil varieties using biotechnological approaches and searching for new effective fungicides, and also in order to devise novel control strategies we have been identifying and characterizing pathogen effectors that play key roles in pathogenicity, which are the potential targets of genetic and chemical control. Objective 1. Evaluate downy mildew resistance of sweet basil generated using biotechnology and understand the molecular basis of basil-Peronospora belbahriiinteractions To generate downy mildew-resistantsweet basil andunderstand the molecular basis of basil-Peronospora belbahriiinteractions, we previously generated T0 transgenic plants overexpressing a putative resistance-related geneOblectin1. During this project period, we determined the transgene expression levels from 16 randomly selected T0 transgenic lines out of a total of 42 using RT-qPCR. We selected four lines with relatively high expression level for further analysis. From their T1 plants, we utilized an approach that combines kanamycin resistance assays with transgene copy number determination via qPCR to identify potential homozygous transgenic T1 plants. For each T0 line, 3-4 potential homozygous transgenic T1 plants were identified and T2 seeds were harvested. Through kanamycin resistance assays of T2 plants, we further identified the homozygous transgenic lines. 2-4 homozygous T2 lines originated from each of four T0 lines have been identified, and will be subjected to disease resistance assays. In addition, to generate downy mildew resistance of sweet basil, previously we performed CRISPR/Cas9-mediated gene editing of two basil candidate susceptibility (S) genesObDMR1andObDMR6, the basil homologs of ArabidopsisDMR1andDMR6, mutation of which conferred nearly complete resistance to Arabidopsis downy mildew pathogenHyaloperonospora arabidopsidis. During the project period, we have obtained the T2 seeds from transgene-free complete knockout T1 mutants for bothObDMR1andObDMR6. For gene editing ofObDMR1, we obtained T2 seeds from homozygous mutant lines derived from three T0 lines. For gene editing ofObDMR6, T2 seeds were harvested from transgene-free complete knockout lines originated from 5 independent T0 lines. We did infection assays in the lab by inoculating the leaves withP. belbahriisporangial suspension, followed by quantification of the pathogen biomass at 4 days post inoculation (dpi) using qPCR and sporangial production at 9 dpi. ForObDMR1mutants, we have not obtained conclusive results as reduced level of disease was observed in one experiment, but not in the second one we have performed. Additional experiments with optimized infection assay conditions are needed to further evaluate these plants. ForObDMR6mutants, we consistently observed significantly reduced pathogen biomass and sporangial production in repeated experiments, suggesting that mutation ofObDMR6enhances downy mildew resistance. Objective 2.Evaluate the efficacy of newly identified anti-oomycete natural metabolites in control ofPhytophthorablight of papaya During this project period, we were not able to work on this objective. Objective 3. IdentifyPhytophthora palmivoraeffectors that play key roles in pathogenesis and the underlying mechanisms During this project period, we have focused on aP. palmivoraeffector, Ppal15kDa. Ppal15kDa was initially identified as a secreted glycoprotein in the culture filtrate ofP. palmivoraby a collaborator from Thailand. We have further determined its role in pathogenicity by overexpressing it inNicotiana benthamiana, and generating its mutants using CRISPR/Cas9-mediated gene editing. We found that transient expression of Ppal15kDa inN.benthamianaby agroinfiltration enhancedP. palmivorainfection. We generated six Ppal15kDa mutants with diverse mutations throughgene editing. All mutants were compromised in pathogenicity onN. benthamianaand papaya, although at varyinglevels. Two mutants with all Ppal15kDa copies mutated almost completely lost pathogenicity. The mutants were also affected indevelopment as they produced smaller sporangia, shorter germ tubes, and fewer appressoria. Altogether, these results suggest that Ppal15kDa plays an important role in normal development ofP. palmivorainfection structures, and pathogenicity. In addition, we found that Ppal15kDa homologs are broadly present inPhytophthoraspp., includingP. megakarya,P. cactorum,P. parasitica(current name:P. nicotianae),P. sojae,P. cinnamomiandP. capsici. As such, Ppal15kDa and homologs may be a promissing target of genetic and chemical control of a range ofPhytophthoraspp. In addition, we have identified two RxLR effectors that are highly induced during infectioncompared with vegetative hyphal growth based on published transcriptomic data. We have performed RT-qPCR to confirm their high level of induction during infection. We have selected one, designated PpalRxLR1, for generating mutants using CRISPR/Cas9 gene editing to determine its role in pathogenicity.

Publications

• Type: Book Chapters Status: Published Year Published: 2020 Citation: Tian, M., Navet, N., and Wu, D. 2020. CRISPR/Cas9-mediated gene editing of the plant pathogenic oomycete Phytophthora palmivora. Book chapter in CRISPR-Cas Methods. Springer Protocols Handbooks https://link.springer.com/book/10.1007%2F978-1-0716-0616-2), Page 87-98.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Navet, N. and Tian, M. 2020. Efficient targeted mutagenesis in allotetraploid sweet basil by CRISPR/Cas9. Plant Direct, 4(6): e00233.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Navet, N. and Tian, M. 2020. Agrobacterium-mediated transformation of sweet basil (Ocimum basilicum) Bio-protocol, 10(22): e3828.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Pettongkhao, S., Navet, N., Schornack, S., Tian, M., and Churngchow, N. 2020. A secreted protein of 15 kDa plays an important role in Phytophthora palmivora development and pathogenicity. Scientific Reports, 10: 2319.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Standish, J.R., Purayannur, S., Bowman, M., Childs, K.L., Tian, M., Quesada, L.M. 2020. Predicting the Peronospora belbahrii secretome for in silico identification of effector proteins. (Abstr.) Phytopathology 110: S1.18. https://doi.org/10.1094/PHYTO-110-7-S1.1
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Standish, J.R., Bowman, M.J., Childs, K.L., Tian, M., Quesada-Ocampo, L.M. 2020. Utilizing comparative genomics to develop species-specific diagnostic markers for basil downy mildew. APS annual meeting 2020 abstract.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Johnson, E.T., Quesada-Ocampo, L., Bowman, M., Childs, K., and Tian, M. 2020. Differential expression of genes encoding sugar transporters in the basil pathogen Peronospora belbahrii. APS annual meeting 2020 abstract.