Functional characterization of Lr67res gene from wheat engineered on rice (Oryza sativa L.) to confer disease resistance against Magnaporthe oryzae

Date

12-2020

Abstract

The rice blast disease caused by the fungus Magnaporthe oryzae is one of the most devastating fungal diseases of rice that leads to significantly reduced rice yield. While resistance (R) genes associated with rice blast resistance are known, only few have been successfully cloned and characterized. There are only limited number of R genes that confer resistance on seedling and adult stages of rice. Therefore, it is indispensable to research on novel utilization strategies for rice blast R genes to establish rice varieties with broad-spectrum and durable resistance against the rice blast disease. The gene Lr67res from Triticum aestivum (wheat) has been shown to be responsible for partial and durable resistance against rust pathogens: Puccinia striiformis f. sp. tritici, Puccinia graminis f. sp. tritici, and powdery mildew pathogen, Blumeria graminis f. sp. tritici on wheat. Lr67res encodes a predicted hexose transporter (LR67res) which reduces glucose uptake through heterodimerization with functional transporters limiting the growth of multiple biotrophic pathogen species on wheat. Lr67res is reported to be transferrable and functional to close relatives of wheat such as barley. However, it has not been investigated on rice yet, but is speculated to have similar effects against the fungal pathogen M. oryzae. In this study, the effects of wheat Lr67res on rice against M. oryzae was investigated. The Lr67res gene from wheat was transferred in rice through Agrobacterium-mediated transformation. Three (3) stable transgenic homozygous Lr67res rice lines were identified through Glucuronidase (GUS) assay and Polymerase Chain Reaction (PCR). However, only two (2) transgenic events were forwarded for subsequent analysis due to limited number of seeds and high mortality rate of the third transgenic line. Spray inoculated transgenic rice at seedling stage exhibited relatively less and smaller lesions compared to those of IR64 wild type. Similar result was observed on punch-inoculated leaf at adult stage of the transgenic plants. Delayed infection rate was observed in post-inoculated panicle through injection method. The infection density of the pathogen determined at seedling stage by quantitative RT-PCR (qRT-PCR) assay showed no significant pattern. Several important agronomic traits of the transgenic plants were assessed relative to the wild type IR64 and showed no significant adverse effects, indicating that the transgene did not cause any detrimental effects on the fitness of the host. The qRT-PCR showed that all the transgenic lines expressed the Lr67res transcript with comparable levels of expression (P < 0.0001) relative to the wild type IR64 on seedling stage. At adult stage, a significant decrease of expression was observed on the transgenic rice (1.25-24.31-fold) relative to the wild type IR64. Fourteen differentially modulated genes were identified through global RNA-seq analysis. Six (6) genes were repressed in the transgenic rice challenged with M. oryzae, including those involved in methyl chloride transferase activity, encryption of a putative protein from epimerase/dehydratase family and HPP family protein, glutathione-S-transferase similar activity, and phosphatidylcholine biosynthetic process. On the other hand, eight (8) genes were upregulated including those associated with jasmonate-regulated myb transcription, encoding of probenazole-inducible protein, malate synthase activity, encrypting of allergen V5/Tpx-1 related family protein, a pathogenesis related gene, trans-membrane protein that is linked to a defense response, a hypothetical protein coding conserved gene, and an asparagine synthetase acting unit. Global RNA-seq analysis revealed that the two generated transgenic lines were not distantly different from each other. With that of wild type IR64 based on the repressed and upregulated genes upon M. oryzae challenge, it is indicated that the insertion of the transgene Lr67res did not recruit some off targets that might have risked the host fitness. The findings in this study show that Lr67res multipathogen resistance gene from wheat is functionally transferrable to rice and confers resistance against M. oryzae at seedling and adult stages. Keywords: Lr67, broad-spectrum resistance, multipathogen resistance, A. tumefaciens- mediated transformation, partial resistance, rice fungal pathogen

Document Type

Dissertation

Degree

Doctor of Philosophy in Microbiology

College

Graduate School (GS)

Adviser/Committee Chair

Rina B. Opulencia

Committee Member

Roberta N. Garcia, Mary Jeanie T. Yanoria, Bo Zhou, Rina B. Opulencia

Language

English

LC Subject

Rice -- Disease and pest resistance -- Genetic aspects, Rice -- Genetic engineering, Rice -- Breeding. Rice -- Diseases and pests -- Control, Rice blast disease, Microbiology Institute of Biological Sciences

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 996 2020 M5 M34

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