Author ORCID Identifier

https://orcid.org/0009-0001-2901-9595

Date Available

8-13-2025

Year of Publication

2025

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Agriculture, Food and Environment

Department/School/Program

Plant Pathology

Faculty

Dr. Mark Farman

Faculty

Dr. Nicole Gauthier

Abstract

Magnaporthe oryzae is a filamentous fungal pathogen that causes destructive diseases in cereals, including rice blast, wheat blast, and gray leaf spot. It is responsible for 10–30% annual yield losses in rice (Nalley et al., 2016) and up to 100% loss in wheat under favorable conditions (Kohli et al., 2011). Disease management typically relies on the use of resistant cultivars, but M. oryzae rapidly overcomes host resistance by mutating or deleting avirulence (AVR) genes recognized by host immune receptors. This rapid adaptation, often occurring within one to two growing seasons, presents a major challenge to breeding for durable resistance.

The wheat blast pathotype (M. oryzae Triticum; MoT) exemplifies this challenge. Limited resistance sources in wheat, coupled with the ineffectiveness of fungicides and the pathogen’s high mutation rate, underscore the urgent need for alternative management strategies. One promising approach is to introduce non-host resistance genes from rice into wheat. The rice resistance gene Piz-t, which encodes an NLR immune receptor, recognizes the fungal effector AvrPiz-t and confers strong resistance (Zhou et al., 2006; Li et al., 2009). Two alleles of AvrPiz-t have been identified in MoT: Br7 and Br80 (Navia, 2022).

To test whether Piz-t could be a viable strategy for wheat blast resistance, I functionally assessed the Br7 and Br80 alleles by expressing them in the M. oryzae strain Guy11, which lacks a functional AvrPiz-t allele. In virulence assays on Piz-t-containing rice cultivars, Br7 triggered an avirulent phenotype, while Br80 produced inconsistent results—some transformants were non-pathogenic, others remained virulent. These outcomes suggest that Br80 is either non-functional or subject to variable expression or silencing, while Br7 retains avirulence function.

Such unexpected phenotypes may result from silencing of the AvrPiz-t transgene or instability of its expression. While transgene silencing is well documented, similar regulatory variation can also affect native AVR genes through epigenetic or expression-level mechanisms.

Following a discussion between Dr. Yulin Jia and Dr. Farman regarding a mixed infection phenotype on Pi9-containing rice, I investigated the M. oryzae isolate SSID116, which carries the endogenous AvrPi9 gene. rkWhen inoculated onto the Pi9-containing line Nipponbare::Pi9, SSID116 occasionally formed rare lesions, despite carrying a functional AVR gene. These mixed phenotypes suggested possible evasion of Pi9-mediated immunity. To test whether AvrPi9 is essential for recognition, I generated a ∆AvrPi9 knockout construct for functional analysis.

To explore whether lesion formation could result from differential expression of AvrPi9, I performed lesion-level RNA-seq on infected Nipponbare::Pi9 tissue. AvrPi9 was detected in all lesions, including rare lesions, at levels comparable to other samples. This indicates that rare lesion formation is not due to absence or silencing of AvrPi9. Importantly, housekeeping gene expression remained stable across all lesions, confirming that differences were not caused by RNA quality or fungal biomass.

These findings support the hypothesis that rare lesion formation may reflect clonal variation in effector gene expression (CVGE). To further explore this, I conducted genome-wide lesion-level RNA-seq to assess variability in overall gene expression, AVR gene expression, and R gene (Pi9) expression. My analysis revealed that while housekeeping genes were consistently expressed, AVR genes—particularly AvrPi9—exhibited significant variability between lesions.

Together, these results highlight lesion-specific variation in effector expression as a potential strategy for immune evasion by M. oryzae. This work provides a foundation for investigating transcriptional plasticity and clonal regulation as novel mechanisms driving pathogen adaptation and resistance breakdown.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2025.384

Funding Information

This study was supported by the Kentucky Science & Engineering Foundation Grant (No. : KSEF-3117-RDE-017) from 2014-2016 and the National Science Foundation Grant (no.:1000100147) from 2018-2022.

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