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Examining the resistance mechanisms and characterizing candidate resistance genes in soybeans against Phytophthora sojae using forward and reverse genetics

Nguyen, Nghi Song
http://orcid.org/0000-0002-7341-7013
http://rave.ohiolink.edu/etdc/view?acc_num=osu1701100168394485

Abstract Details

2023, Doctor of Philosophy, Ohio State University, Plant Pathology.
Phytophthora root and stem rot is one of the top ten most yield-limiting soybean [Glycine max (L.) Merr] diseases in the U.S. and Canada. The causal agent of this disease, Phytophthora sojae (Kaufmann & Gerdemann), is an oomycete (water mold) organism that is often managed by using disease-resistant soybean cultivars containing a single Rps gene to a specific pathotype of the pathogen combined with quantitative disease resistance (QDR). Due to the limitation of the number of effective Rps genes available, as the populations of this pathogen have adapted to these genes, it is a necessity to find new Rps genes and identify perfect markers for QDR genes, which have a smaller effect but can resist all pathotypes of P. sojae, that can be used in the development of modern soybean cultivars. In this dissertation, the main approach for all three chapters is to examine the mechanisms of disease resistance in soybeans towards P. sojae using different forward and reverse genetic approaches. A previous study found more than 100 candidate genes by mapping the expression quantitative disease-resistance loci (eQDRL) from the Conrad × Sloan population. To explore and validate the functions of these genes, a fast neutron (FN) population from the University of Minnesota, derived from soybean cultivar M92-220, with these genes deleted was employed. Thus, the first chapter’s first objective was to compare cultivars M92-220 and Conrad at phenotypic and transcriptomic levels for their QDR resistance to determine if the molecular mechanisms associated with the eQDRLs were similar to those previously found in Conrad. Then, the next objective was to explore how the loss of the candidate genes using mutants that were highly susceptible would affect the soybean resistance response following inoculation with P. sojae by examining the three most susceptible FN mutants. Conrad and M92-220 were found to share high levels of QDR in the phenotypic assay as well as having several similar defense-related pathways enriched in the transcriptomic study such as phenylpropanoid and flavonoid biosynthesis, MAPK signaling, cell wall modification, and plant-pathogen interaction. However, there were also a few uniquely enriched pathways including stilbenoid, diarylheptanoid, and gingerol biosynthesis, arachidonic acid metabolism, and monobactam biosynthesis found only in M92-220 but not in Conrad that can be explored as potential new mechanisms of resistance towards P. sojae for future QDR studies. In addition, transcriptomic comparison of FN mutants to M92-220 identified potentially important KEGG pathways at either one or both time points examined, including sesquiterpenoid and triterpenoid biosynthesis, thiamine metabolism, arachidonic acid, stilbenoid, diarylheptanoid and gingerol biosynthesis, and monobactam biosynthesis that could be due to the effect of deleted genes. Moreover, there were ten genes differentially expressed during P. sojae infection in M92-220 that were not expressed in one or more of the moderately susceptible mutants of the mutants. This validated the involvement of some of the genes from previous expression quantitative disease resistant loci (eQDRL) studies in QDR resistance mechanism in soybeans and provide some indication of what role they may play. The results of this study provided evidence for several known and potential novel QDR pathways as well as candidate genes that can be used for further functional studies in soybeans. In the second chapter, a functional study using two different gene silencing approaches was done for a MORC family CW-type zinc finger coiled-coil domain protein 3 transcription factor gene (Glyma.01g156800) found near an eQDRL hot spot (Satt198) on chromosome 1 found in the same eQDRL mapping study mentioned above. The objective of this study was to characterize the function of this gene in resistance against P. sojae using Agrobacterium rhizogenes-mediated transformation and Cowpea severe mosaic virus-mediated gene silencing. Inoculation of P. sojae isolate OH25 on cultivar Conrad with known QDR using both of these gene silencing methods showed a reduction in resistance by the greater size of lesions following inoculation when gene Gmzf-CW3 was silenced compared to the non-silenced groups. However, for confirmation of the gene expression study using RT-qPCR, gene silencing occurred in only the Agrobacterium-mediated transformation. Additionally, Gmzf-CW3 was only expressed in soybean roots, which provided evidence for its involvement in the QDR of soybeans as loss of resistance occurred when the gene was silenced in roots but not in stem inoculation. The results suggest that the MORC family CW-type transcription factor Gmzf-CW3 gene may play a role in the complex QDR mechanism in the highly resistant cultivar potentially by regulation of epigenetic regulation and protein-protein interaction. The third chapter examined a putative candidate Rps-mediated resistant gene found in a previous Ren-Seq study of 34 soybean genotypes. This gene was found in the Rps1c locus and encoded for nucleotide-binding, leucine-rich repeat (NB-LRR) proteins. The objective of this chapter was to examine the loss of function in the resistant cultivar Williams 79 which contains Rps1c when inoculated with P. sojae isolate OH1 using the CPSMV-mediated gene silencing method. The increased susceptibility of Williams 79 in gene-silenced groups compared to non-silenced groups indicates this gene may function as an Rps1c candidate gene although the silenced genotype was not confirmed in the gene expression assay using RT-PCR, which may be due to incomplete gene silencing.
Feng Qu (Advisor)
Anne Dorrance (Committee Member)
Guo Liang Wang (Committee Member)
Xia Ye (Committee Member)
Leah McHale (Committee Member)
231 p.
Plant Biology; Plant Pathology; Plant Sciences
Soybeans, Phytophthora sojae, comparative transcriptomics, disease resistance genes, host resistance, host pathogen interactions, gene silencing, forward genetics, reverse genetics.

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Citations

  • Nguyen, N. S. (2023). Examining the resistance mechanisms and characterizing candidate resistance genes in soybeans against Phytophthora sojae using forward and reverse genetics [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1701100168394485

    APA Style (7th edition)

  • Nguyen, Nghi. Examining the resistance mechanisms and characterizing candidate resistance genes in soybeans against Phytophthora sojae using forward and reverse genetics. 2023. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1701100168394485.

    MLA Style (8th edition)

  • Nguyen, Nghi. "Examining the resistance mechanisms and characterizing candidate resistance genes in soybeans against Phytophthora sojae using forward and reverse genetics." Doctoral dissertation, Ohio State University, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=osu1701100168394485

    Chicago Manual of Style (17th edition)

osu1701100168394485
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This open access ETD is published by The Ohio State University and OhioLINK.