Progress 10/01/14 to 09/30/19
Outputs
Target Audience:Plant scientists working on both basic and translational research, and agricultural industries. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training and professional development opportunities for three graduate students (one Ph.D. student and one M.S. graduated, one M.S. student current), six undergraduate students, and a scientist who is the PI of this project. How have the results been disseminated to communities of interest?The results have been disseminated to the scientific communities through presentations in American Phytopathological Society annual meetings, Oomycete Molecular Genetics Network annual meetings, International Society for Molecular Plant-Microbe Interactions Congress, CTAHR Student Symposium, and journal articles. Part of the results was also disseminated to interested scientists through informal oral and Email communications. 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) To investigate the potential of recently published SAR-inducing molecules for use in control of basil downy mildew. Two commercially available SAR-inducing molecules azelaic acid and pipecolic acid were tested for their efficacy in control of basil downy mildew. Both chemicals did not provide basil plants with effective protection against the downy mildew disease. To discover additional compounds that are highly efficient in controlling basil downy mildew with low/no toxicity and diverse modes of action, we tested a natural metabolite library of 800 small molecules with diverse structures and chemical properties. Due to the small amount of each chemical and the large number of the chemicals, it was not practical to directly test their efficacy using plant infection assays. As the obligate biotroph Peronospora belbahrii can't grow in media, we developed a high-throughput screening method using another oomycete pathogen Phytophthora palmivora. By using this method, we identified 18 plant-derived compounds with low risk to environments and human health and obtained them in a larger quantity. The high-level anti-oomycete activities were confirmed for 10 of them using P. palmivora mycelium growth assays in media. We further tested the anti-fungal/oomycetes activities of these compounds against 3 other agriculturally important pathogens, including the oomycete pathogen Phytophthora colocasiae and fungal pathogens Colletotrichum gloeosporioides and Corynespora cassicola use mycelium growth assays in media. 7 of them exhibited very high and broad-spectrumactivities. We have tested 4 compounds for their efficacy to control basil downy mildew. All four significantly delayed disease onset and sporulation when sporangia were inoculated in the presence of the compounds. However, when we applied the compounds 5-7 days post inoculation, or sprayed them 24 h or 48 h before inoculation, we did not observe significant disease control effect. Despite that these chemicals are not effective against P. belbarhii, they may be more effective in controlling other plant pathogenic oomycetes. 2) To dissect the pathogenesis mechanisms of basil downy mildew pathogen Peronospora belbahrii to develop targeted chemical and/or genetic control strategies. To understand the pathogenesis mechanisms of Peronospora belbahrii, we generated a set of genomic sequence data for identifying candidate virulence genes. We sequenced the transcriptomes of purified P. belbahrii sporangia, non-infected basil leaves, and infected basil leaves during the infection time course. By using bioinformatics approaches, we identified the secretome, putative effector proteins and their expression patterns during infection. A dozen P. belbahrii effector candidate (PbEC) genes that likely play key roles in infection were selected for detailed experimental characterization.Six PbEC genes were induced during infection, with PbEC1 and PbEC2 highly induced. The signal peptides of PbEC1 and PbEC2 were functionally validated using a yeast secretion assay, suggesting that they are bona fide effectors that function inside the host cell. We next sought to determine the roles of PbECs in pathogenesis. Initially we attempted to utilize Agrobacterium-mediated transient expression assay followed by pathogen infection. We were able to transiently expressing PbEC2 and its hairpin RNA (hpRNA)-forming construct in basil leaves. To be able to quantify the disease at the early infection stage, we established a sensitive quantitative PCR (qPCR) approach, which utilizes the primers amplifying the internal transcribed spacer 2 (ITS2) region of P. belbahrii, to quantify pathogen growth. However, even with the sensitive disease quantification method, determining the roles of PbEC2 using transient overexpression and host-induced gene silencing (HIGS) did not reliably generate reproducible results. Some of the potential reasons could be the expression of transgene is not high enough and/or too transient. Next we performed HIGS through stable transformation. We targeted PbEC1, PbEC2 and an RxLR-containing endoplasmic reticulum resident protein PbORCER1 to evaluate whether HIGS functions in basil-P. belbahrii interactions, determine the roles of these genes in pathogenesis, and potentially generate disease resistant plants. We optimized a system for effective transformation of sweet basil. With this system, we generated 40, 13, and 39 independent transgenic T0 plants that express hpRNAs for HIGS of PbEC1, PbEC2 and PbORCE1 respectively. 4-5 independent lines with higher level of transgene expression for each gene were selected for further analysis. Homozygous transgenic plants in the successive generations were selected by performing a kanamycin resistance assay coupled with a quantitative polymerase chain reaction (qPCR) on the segregating populations. For PbEC1 and PbORCER1, we were able to obtain T2 homozygous transgenic plants derived from 3-4 independent T0 transgenic lines. Upon infection with P. belbahrii, the expression of PbORCER1 on homozygous transgenic plants expressing hpRNA of PbORCER1 was significantly reduced compared with its expression on the wild type (WT) plants, suggesting successful silencing of PbORCER1. Knockdown of PbORCER1 expression corresponded to the reduced pathogen growth, indicating the role of PbORCER1 in pathogen proliferation. The pathogen growth on transgenic plants expressing hpRNA of PbEC1 was also significantly reduced compared to WT. However, the silencing of PbEC1 transcripts was not observed likely because its expression was very highly induced during infection, which masked the detection of silencing. Collectively, our data suggest that HIGS is functional in silencing P. belbahrii genes, and therefore holds great potential for gene function analyses and controlling basil. However, these results need be further validated with repeated infection assays. To identify host factors involved in basil downy mildew disease development, we used CRISPR/Cas9-mediated gene editing technology to generate mutations in two basil genes ObDMR1 and ObDMR6, which are the homologs of Arabidopsis DMR1 and DMR6. Arabidopsis DMR1 and DMR6 and their homologs in multiple other plant species were identified as Susceptibility (S) genes that are required for infection of multiple fungal/oomycete pathogens. We hypothesized that their homologs in basil play roles in downy mildew disease development. By targeting ObDMR1, we established an efficient gene editing system for sweet basil. We used Agrobacterium-mediated transformation to express gene editing reagents. Mutations were detected in 92.6% of 54 T0 transgenic lines, with one of them as a complete knockout mutant. ObDMR1 homozygous mutants with some being transgene-free were identified from T1 segregating populations. This study demonstrated the feasibility of the application of CRISPR/Cas9 in basil functional genomics and breeding. Initial pathogen infection assays did not give us a conclusive result on whether these mutant lines significantly enhance resistance against P. belbahrii. For targeted mutagenesis of ObDMR6, a total of 71 transgenic lines were obtained. Mutations were observed in 41 plants out of 44 analyzed. Complete mutation of all copies of ObDMR6 was observed in 12 T0 transgenic plants. We have identified transgene-free complete knockout mutants in T1 generation. These plants are currently growing for seeds, which will be used for infection assays. In summary, we have established various resources and tools to dissect the molecular basis of basil-P. belbahrii interactions and breed for downy mildew resistance and other beneficial traits. In addition, some of the transgenic lines or transgene-free gene-edited lines with increased resistance has the potential to be used in sweet basil production.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Navet, N. and Tian, M. 2019. Targeted mutagenesis of basil candidate susceptibility gene DMR1 using CRISPR/Cas9. (Abstr.) Phytopathology 109: S2.22. https://doi.org/10.1094/PHYTO-109-10-S2.22
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2019
Citation:
Navet, N. 2019. Dissecting the molecular basis of basil-Peronospora belbahrii interactions and genetic engineering for disease resistance. PhD Dissertation, University of Hawaii at Manoa.
- Type:
Book Chapters
Status:
Awaiting Publication
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 Nature (in press).
- Type:
Journal Articles
Status:
Accepted
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 Report.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2020
Citation:
Navet, N. and Tian, M. 2020. Efficient targeted mutagenesis in allotetraploid sweet basil by CRISPR/Cas9. Plant Biotechnology Journal.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Standish, J.R., Purayannur, S., Bowman, M., Childs, K., Tian, M., and Quesada-Ocampo, L.M. 2020. Predicting the Peronospora belbahrii secretome for in silico identification of effector proteins. 97th Annual meeting of American Phytopathological Society Southern Division. Charleston, South Carolina. Feb 9-12, 2020.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Plant scientists working on both basic and translational research, and agricultural industries. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training and professional development opportunities for two graduate students, two undergraduate students, and an assistant researcher who is the PI of this project. How have the results been disseminated to communities of interest?The results have been disseminated to the scientific communities through presentations in CTAHR Student Symposium, and an open-access journal article. Part of the results was also disseminated to interested scientists through informal oral and Email communications. What do you plan to do during the next reporting period to accomplish the goals?For Objective 1: We will test four selected plant-derived natural metabolites with high anti-fungal/oomycete activities on diseases caused by other oomycete pathogens such as Phytophthora palmivora and P. colocasiae, both of which cause significant losses on tropical crops grown in Hawaii. This plan is not closely related to basil downy mildew, which is the subject of this HATCH project. However, as the chemicals were identified through this project, we are intrigued and obligated to see whether they can be utilized to control other diseases of agricultural importance. For Objective 2: To determine the role of three candidate effector genes in P. belbahrii pathogenesis, we will obtain T2 homozygous transgenic seeds expressing dsRNAs of these genes and perform further detailed characterization. The accumulation of pathogen gene-targeting small RNAs, level of pathogen gene silencing and altered disease during infection will be tested. For the identification of host factors involved in basil downy mildew disease development, we will obtain seeds from homozygous/biallelic mutants of ObDMR1, which will be planted and subjected to disease infection assay. We will continue to generate transformants and identify the gene-edited mutants for ObDMR6.
Impacts
What was accomplished under these goals?
1) To investigate the potential of recently published SAR-inducing molecules for use in control of basil downy mildew. During the last reporting period, we tested four plant-derived natural metabolites, which exhibited high anti-oomycete activities when tested for inhibition of in vitro growth of the oomycete pathogen Phytophthora palmivora and P. colocasiae, and suppressed basil downy mildew disease onset and sporulation when they were applied by mixing with sporangia, for their curative and preventive effects to control basil downy mildew. Unfortunately, their effect in controlling basil downy mildew was not significant. We did not do further work in this period. 2) To dissect the pathogenesis mechanisms of basil downy mildew pathogen Peronospora belbahrii to develop targeted chemical and/or genetic control strategies. To dissect the pathogenesis mechanisms of basil downy mildew pathogen Peronospora belbahrii, we have been characterizing the putative effector proteins, which are hypothesized to play important roles in pathogenicity based on research with other fungal/oomycete pathogens. Based on expression patterns during infection and validation of the functionality of signal peptides, we have selected 3 candidate effector genes PbEC1, PbEC2 and PbORCE1 to test whether host-induced gene silencing (HIGS) functions in silencing P. belbahrii genes during infection and determine their roles in disease development using HIGS. Continuing from the last reporting period, we have generated stable transgenic plants expressing dsRNAs that target all these 3 genes. 13 independent transgenic T0 plants that express PbEC2 hairpin-forming construct, and over 30 independent transgenic T0 plants for HIGS of PbEC1 and PbORCE1 have been obtained. T1 seeds have been harvested for these plants. To select transgenic lines for further analyses, we determined the expression level of dsRNAs in T0 plants using reverse transcription quantitative PCR (RT-qPCR). Five independent lines with relatively high level of expression were selected for each gene for further analyses. To identify homozygous transgenic plants in the T1 generation, we developed an assay involving the use of antibiotics selection followed by quantitative PCR (qPCR) to determine the transgene zygosity. Putative homozygous T1 plants have been identified for each selected line and now growing to produce homozygous T2 seeds. Once the seeds are harvested, they will be used for infection assay to determine the silencing of these pathogen genes and infer their roles in disease development based on the altered disease level. To identify host factors involved in basil downy mildew disease development, we have begun to use CRISPR/Cas9-mediated gene editing technology to generate mutations in two basil genes ObDMR1 and ObDMR6, which are the homologs of Arabidopsis DMR1 and DMR6. Arabidopsis DMR1 and DMR6 were identified as Susceptibility (S) genes that are required for infection of Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis. Their mutations in Arabidopsis confer nearly complete resistance to H. arabidopsidis. In addition, silencing of their homologs in other plant species have been reported to mediate resistance to fungal/oomycete pathogens. We hypothesized that their homologs in basil play roles in downy mildew disease development. Generating mutants of these two genes using CRISPR/Cas9 not only allows us to analyze their function in disease development, but also generate novel sweet basil disease resistant varieties. Editing S genes using CRISPR/Cas9 technology has been shown to be a fast and effective way to breed for disease resistance. To design single guide RNAs (sgRNAs) that exactly match the sequences of the target genes in sweet basil cultivar Genovese, which will be used for transformation and genome editing, the genomic DNA sequences of both genes were amplified from Genovese and sequenced. sgRNAs have been designed and cloned to the plant binary vector pKSE401, which was developed for CRISPR/Cas9-mediated gene editing in dicots. For each gene, two constructs expressing either one sgRNA or two sgRNAs were generated. Transgenic plants have been obtained for editing of ObDMR1, 24 and 7 T0 transgenic plants were obtained for the 1-sgRNA and 2-sgRNA construct respectively. Mutations were detected in 15 transgenic plants with 6 harboring heterozygous mutations and 10 containing chimeric mutations. The T0 plants with heterozygous mutations are growing for T1 seeds, from which homozygous mutants will be identified. Meanwhile, more T0 transgenic plants will be generated to identify the mutants with homozygous or biallelic mutations in T0 generations. Seeds generated from plants with homozygous or biallelic mutations will be planted and subjected to plant infection assay.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Shao, D. and Tian, M. 2018. A qPCR approach to quantify the growth of basil downy mildew pathogen Peronospora belbahrii during infection. Current Plant Biology, 2018, 15:2-7.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Navet, N., Shao, D., Dose, H., and Tian M. 2018. Engineering basil plants for resistance against downy mildew. CTAHR Student Symposium, University of Hawaii at Manoa, Honolulu, HI.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Dose, H., Navet, N., and Tian, M. 2018. Application of CRISPR/Cas9 gene editing technology in generating sweet basil resistance to downy mildew. CTAHR Student Symposium, University of Hawaii at Manoa, Honolulu, HI.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Plant scientists working on bothbasic and applied research,and agricultural industries. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training and professional development opportunities for two graduate students, one undergraduate student, and an assistant researcher who is the PI of this project. How have the results been disseminated to communities of interest?The results have been disseminated to the scientific communities through a master's thesis and a presentation at Oomycete Molecular Genetics Network (OMGN) annual meeting held at Asilomar, California. Part of the results was also disseminated to interested scientists through informal oral and Email communications. What do you plan to do during the next reporting period to accomplish the goals?For Objective 1,We will test the control efficacy of the four selected plant-derived natural metabolites with high anti-fungal/oomycete activities on diseases caused by other oomycete pathogens such as Phytophthora palmivora and P. colocasiae, both of which cause significant losses on tropical crops grown in Hawaii. This plan is not closely related to basil downy mildew, which is the subject of this HATCH project. However, as the chemicals were identified through this project, we are intrigued and obligated to see whether they can be utilized to control other diseases of agricultural importance. For Objective 2,We will obtain homozygous transgenic plants expressing hairpin-forming construct of PbEC2 and perform detailed characterization. The expression of dsRNAs, accumulation of PbEC2-targeting small RNAs, silencing of PbEC2 and altered disease during infection will be tested. We will continue to characterize PbEC1 and PbORCE1 by obtaining the transgenic plants for HIGS and performing characterization as for PbEC2. We will also continue to characterize the role of the PbORCE1 homolog in pathogenesis using P. palmivora.
Impacts
What was accomplished under these goals?
1) To investigate the potential of recently published SAR-inducing molecules for use in control of basil downy mildew. During the last reporting period, we selected and tested four plant-derived natural metabolites for their efficacy to control basil downy mildew. All four significantly delayed disease onset and sporulation when sporangia were inoculated in the presence of the compounds. However, when we applied the compounds 5-7 days post inoculation, we did not observe significant disease control effect, suggesting their poor curative properties. During the current project period, we tested their preventive propertiesby spraying the compounds 24 h or 48 h before pathogen inoculation. Unfortunately, the disease control effect was not significant. 2) To dissect the pathogenesis mechanisms of basil downy mildew pathogen Peronospora belbahrii to develop targeted chemical and/or genetic control strategies. To dissect the pathogenesis mechanisms of basil downy mildew pathogen Peronospora belbahrii, we have been characterizing the putative effector proteins, which are hypothesized to play important roles in pathogenicity based on research with other fungal/oomycete pathogens. During the previous reporting period, we did gene expression analyses of 12 P. belbahrii effector candidate (PbEC) genes during infection and established a sensitive method to quantify pathogen biomass for evaluating the function of effector genes in disease development. Following that, we started to use PbEC2, an RxLR effector that is highly induced during infection, to test whether host-induced gene silencing (HIGS) functions in silencing P. belbahrii genes during infection of basil plants using both transient expression and stable transformation. During the current project period, we expressed the hairpin-forming construct of PbEC2 using an Agrobacterium-mediated transient expression assay and then evaluated whether the disease was altered. However, we did not observe conclusive results from the transient assay maybe because the expression level of dsRNAs was low and/or transient. In parallel, we have established the protocol for basil transformation and successfully generated transgenic basil plants expressing PbEC2 hairpin-forming construct for HIGS. We obtained 13 independent transgenic T0 plants, and confirmed the integration of the construct into the basil genome and the expression of hairpin RNAs of PbEC2 using PCR and qPCR. The T1 segregating plants have been growing for T2 seeds, which will be tested for homozygous lines. The homozygous lines will be tested for the silencing of PbEC2 through HIGS and the altered disease. Our initial results with T1 segregating plants of 2 independent transgenic lines showed that the presence of the transgene was associated with the reduced disease. However, this need be confirmed with homozygous lines. In addition, we have been characterizing another two candidate effectors PbEC1 and PbORCE1. PbEC1 was highly induced during infection and appears to encode a very intriguing protein. It has a predicted signal peptide and a C-terminal domain that shares homology with conserved domains present in proteins involved in DNA synthesis and repair (Conserved Protein Domain Family accessions PRK12323 and TIGR00601), suggesting that PbEC1 may contribute to pathogenesis by entering the plant nuclei to interfere with activities related to DNA synthesis and repair of basil plants. PbORCE1 has a putative signal peptide followed by a canonical RxLR effector translocation motif. The orthologs of PbORCE1 were found to be present and highly conserved in all plant pathogenic oomycetes with genomic sequence information available. Identification of its role in pathogenesis may help identify the common pathogenesis mechanisms of oomycete pathogens, many of which are very destructive and severely threaten agricultural production and natural ecosystems. We generated hairpin-forming constructs for both genes and are currently generating transgenic lines. For determining the role of PbORCE1, we also utilized another oomycete pathogen Phytophthora palmivora, which is cultivable with genetic modification tractable. We have shown that overexpression of ORCE1 in P. palmivora enhances pathogen virulence. Currently, we are generating the mutants using CRISPR/Cas9 mediated gene editing via Agrobacterium-mediated transformation, a system that was recently developed by Tian lab and shown to be very effective in editing genes of P. palmivora.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Gumtow, R., Wu, D., Uchida, J., and Tian, M. 2017. A Phytophthora palmivora extracellular cystatin-like protease inhibitor targets papain to contribute to virulence on papaya. Mol Plant Microbe Interact. 2017 Oct 25. doi: 10.1094/MPMI-06-17-0131-FI. [Epub ahead of print]
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2017
Citation:
Shao, D. 2017. Functional characterization of putative effector genes of basil downy mildew pathogen Peronospora belbahrii. 2017, Master Thesis in Tropical Plant Pathology, University of Hawaii at Manoa. Honolulu, HI.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Navet, N., Wu, D., Shao, D., and Tian M. 2017. Functional characterization of a cytoplasmic effector gene highly conserved in plant pathogenic oomycetes. OMGN annual meeting, Asilomar, CA.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Scientists working on both basic and applied research, extension agents and agricultural industries. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training and professional development opportunities for 2 graduate students, 1 undergraduate student, and an assistant researcher who is the PI of this project. How have the results been disseminated to communities of interest?The results have been disseminated to the scientific communities through presentations atInternational Society for Molecular Plant-Microbe Interactions (IS-MPMI) XVII Congress at Portland, Oregon, and CTAHR Student Symposium at University of Hawaii at Manoa. The results have also been disseminated to a biotechnology company through the collaboration with Intrexon. What do you plan to do during the next reporting period to accomplish the goals?For Objective 1,We will continue to test the natural metabolites with broad-spectrum anti-fungal/oomycetes activities for their efficacy to control basil downy mildew. Various concentrations and application times before or post inoculation will be trialed. For Objective 2,Transgenic plants expressing hairpin-forming construct of PbEC2 will be characterized for the expression of dsRNA, accumulation of PbEC2-targeting small RNAs, silencing of PbEC2 and altered disease during infection. If successful, more effector candidate genes will be studied to determine their virulence functions.
Impacts
What was accomplished under these goals?
1. To investigate the potential of recently published SAR-inducing molecules for use in control of basil downy mildew. During the last reporting period, we identified 10 plant-derived natural metabolites with anti-oomycete activity. We further tested the anti-fungal/oomycetes activities of these compounds against 3 other agriculturally important pathogens, including the oomycete pathogen Phytophthora colocasiae and fungal pathogens Colletotrichum gloeosporioides and Corynespora cassicola use mycelium growth assays in media. 7 of them exhibited very high and broad-spectrumactivities. We have tested 4 compounds for their efficacy to control basil downy mildew. All four significantly delayed disease onset and sporulation when sporangia were inoculated in the presence of the compounds. However, when we applied the compounds 5-7 days post inoculation; we did not observe significant disease control effect, suggesting their poor curative properties. We are currently testing their disease preventive properties. 2) To dissect the pathogenesis mechanisms of basil downy mildew pathogen Peronospora belbahrii to develop targeted chemical and/or genetic control strategies. We selected a dozen P. belbahrii effector candidate genes that likely play key roles in infection based on transcripomic data generated in the previous year for further characterization. These candidate genes encode predicted secreted proteins with a translocation motif RxLR (or RxLR-EER) and/or nuclear localization signals (NLS). First, we determined their gene expression patterns during infection using reverse transcription quantitative PCR. Five genes were shown to be induced during infection and the functionality of their predicted signal peptides was confirmed using a yeast invertase secretion system, suggesting that these genes likely encode bona fide effectors that play significant roles in manipulating host cellular processes to cause disease. In parallel, we have been establishing systems to experimentally determine the roles of these genes in pathogenicity through overexpression and host-induced gene silencing (HIGS) via transient expression or stable transformation. Transient gene assays are convenient alternatives to stable transformation for rapid analysis of gene functions. However, the expression may only last a few days. On the other hand, it usually takes 5-7 days for basil downy mildew disease symptoms to develop after inoculation. To be able to determine the function of effector candidate genes using transient assays, a sensitive method to quantify pathogen growth before disease symptoms appear is critical. To this end, we established a very sensitive qPCR method using P. belbahrii-specific primers targeting the internal transcribed spacer region of the nuclear ribosomal DNA. Using PbEC2, an RxLR effector that is highly induced during infection, we are testing whether HIGS functions in silencing P. belbahrii genes during infection of basil plants using both transient expression and stable transformation. Transgenic plants expressing hairpin-forming construct of PbEC2 have been generated and await further characterization.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Wu, D., Navet, N., Liu, Y., Uchida, J., and Tian, M. 2016. Establishment of a simple and efficient Agrobacterium-mediated transformation system for Phytophthora palmivora. BMC Microbiology, 2016, 16: 204
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Shao, D. and Tian, M. 2016. P9-305: Functional characterization of putative effector genes of basil downy mildew pathogen Peronospora belbahrii. International Society for Molecular Plant-Microbe Interactions (IS-MPMI) XVII Congress, 2016, Portland, Oregon.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Wu, D., Navet, N. and Tian, M. 2016. P9-312: Establishment of a simple and efficient agrobacterium-mediated transformation system for Phytophthora palmivora. International Society for Molecular Plant-Microbe Interactions (IS-MPMI) XVII Congress, 2016, Portland, Oregon.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience: Scientists working on both basic and applied research, extension agents and agricultural industries. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training and professional development opportunities for a graduate student and an assistant researcher who is the PI of this project. How have the results been disseminated to communities of interest?The results have been disseminated to the scientific communities through oral and poster presentations in Oomycete Molecular Genetics Network and American Phytopathological Society annual meetings and the CTAHR/Tokyo University of Agriculture and Technology (TUAT) meeting. Knowledge of the applied aspects derived from this project was disseminated to Hawaii basil growers and participants of GoFarm Hawaii training program through an oral presentation in "Confronting Downy Mildew of Basil in Hawaii" workshop. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: The selected natural metabolites that show anti-oomycete activities will be tested for their efficacy to control basil downy mildew in greenhouses. Objective 2: As P. belbahrii is an obligate biotroph, function genomics studies through direct genetic modification of the pathogen is impractical. Since host-induced gene silencing (HIGS) is demonstrated to be a valuable tool to dissect the biological roles of genes from other obligate biotrophic pathogens, we will test whether HIGS functions in silencing P. belbahrii genes during infection of basil plants using 1-2 selected candidate effector genes. Both transient expression and stable transgenic expression through basil transformation will be performed. The effectiveness of HIGS in silencing P. belbahrii genes will be determined. The establishment of HIGS system during the next reporting period is critical in studying virulence functions of many other P. belbahrii genes in the future.
Impacts
What was accomplished under these goals?
Objective 1. To investigate the potential of recently published SAR-inducing molecules for use in control of basil downy mildew. Two commercially available SAR-inducing molecules azelaic acid and pipecolic acid were tested for their efficacy in control basil downy mildew. Both chemicals did not seem to provide basil plants with effective protection against the downy mildew disease. To discover additional compounds that are highly efficient in controlling basil DM with low/no toxicity and diverse modes of action, we obtained a natural metabolite library of 800 small molecules with diverse structures and chemical properties. Due to the small amount of each chemical and the large number of the chemicals, it is not practical to directly test their efficacy using plant infection assays. As the obligate biotroph Peronospora belbahrii can't grow in media, we developed a high-throughput screening method using another oomycete pathogen Phytophthora palmivora. By using this method, 59 chemicals were found to effectively inhibit either spore germination and/or mycelium growth. Among them, 18 plant-derived compounds with low risk to environments and human health were selected and obtained in a larger quantity. The high-level anti-oomycete activities were confirmed for 10 of them using P. palmivora mycelium growth assays in media. 2. To dissect the pathogenesis mechanisms of basil downy mildew pathogen Peronospora belbahrii to develop targeted chemical and/or genetic control strategies. To understand the pathogenesis mechanisms of Peronospora belbahrii, we generated a set of genomic sequence data for identifying candidate virulence genes. We sequenced the transcriptomes of purified P. belbahrii sporangia, non-infected basil leaves, and infected basil leaves during the infection time course. By using bioinformatics approaches, we identified the secretome, putative effector proteins and their expression patterns during infection. A dozen candidate effectors that likely play key roles in infection were selected for detailed experimental characterization. In parallel with the above efforts, we have developed an agroinfiltration-mediated transient expression system in basil for functional genomic studies of P. belbahrii genes. These resources are expected to better our understanding of the molecular basis of basil-P. belbahrii interactions and in turn help design novel disease control strategies.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Wu, D., Win, J., Shao, D., and Tian, M. 2015. Poster 789-P: Dissecting the molecular basis of basil-Peronospora belbahrii interactions. American Phytopathological Society (APS) annual meeting, 2015, Pasadena, California.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Tian, M., Wu, D., and Shao, D. 2015. Oral presentation: De novo assembly and analysis of transcriptome of Peronospora belbahrii. OMGN annual meeting, 2015, Asilomar, California.
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