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Rice blast, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most problematic diseases for rice production, threatening global food security. Genetic resistance to some M. oryzae races can be achieved using major resistance genes that recognize their corresponding fungal avirulence genes. Weedy rice, a close relative of cultivated rice that competes with the crop, has evolved unique genetic mechanisms to resist M. oryzae infections; thus, weedy rice can serve as an excellent resource for blast control. In this study, we assessed disease scores of 183 F₅and F₆recombinant inbred lines (RILs) derived from a weedy rice × crop biparental mapping population and their parental lines, a Black Hull Awn weedy rice strain (PI 653413, RR14) and the aus-196 rice variety, using four distinct common U.S. blast races (IB33, IG1, IE1K, and IC17) under greenhouse conditions. All the parental lines were resistant to all blast races; however, RILs showed a wide degree of variation in resistance. Genotyping-by-sequencing of the RIL population and parents generated 1,498 single-nucleotide polymorphisms, which were used to construct a linkage map, and quantitative trait locus (QTL) mapping of blast resistance was performed using r/qtl. A single major blast resistance QTL on chromosome 12 was mapped to the Pi-ta/Pi39(t)/Ptr locus. Identification of Pi-ta/Pi-39(t)/Ptr as the key contributor to blast resistance in weedy rice provides insight into the evolution and adaptation of weedy rice and can aid in the development of blast-resistant rice varieties through marker-assisted selection. 

Abstract One of the common mechanisms to trigger plant innate immunity is recognition of pathogen avirulence gene products directly by products of major resistance (R) genes in a gene for gene manner. In the USA, theRgenes,Pik-s, PiKh/m, andPi-ta, Pi-39(t), andPtrgenes have been effectively deployed to prevent the infections ofM. oryzaeraces, IB49, and IC17 for some time.Pi-9is only recently being deployed to provide overlapped and complimentary resistance toMagnaporthe oryzaeraces IB49, IC17 and IE1k in the USA. Pi-ta, Pi-39(t), Pi9 are major nuclear binding site-leucine rich (NLR) proteins, and Ptr is an atypical R protein with 4 armadillo repeats. AlphaFold is an artificial intelligence system that predicts a protein 3D structure from its amino acid sequence. Here we report genome sequence analyses of the effectors and avirulence (AVR) genes,AVR-PitaandAVR-Pik, andAVR-Pi9, in 3 differentialM. oryzaeraces. Using AlphaFold 2 and 3 we find strong evidence of direct interactions of products of resistance genesPi-taandPikwithM. oryzaeavirulence (AVR) genes,AVR-PitaandAVR-Pikrespectively. We also found that AVR-Pita interacts with Pi-39(t) and Ptr, and Pi9 interacts with both AVR-Pi9 and AVR-Pik. Validation of direct interactions of two pairs of R and AVR proteins supported a direct interaction mechanism of plant innate immunity. Detecting interaction of both Ptr and Pi39(t) with AVR-Pita, and Pi-9 with both AVR-Pi9 and AVR-Pik, revealed a new insight into recognition of pathogen signaling molecules by these host R genes in triggering plant innate immunity. 

Rice resistance (R) genes have been effectively deployed to prevent blast disease caused by the fungal pathogen Magnaporthe oryzae, one of the most serious threats for stable rice production worldwide. Weedy rice competing with cultivated rice may carry novel or lost R genes. The quantitative trait locus qBR12.3b was previously mapped between two single nucleotide polymorphism markers at the 10,633,942-bp and 10,820,033-bp genomic positions in a black-hull-awned (BHA) weed strain using a weed-crop-mapping population under greenhouse conditions. In this study, we found a portion of the known resistance gene Ptr encoding a protein with four armadillo repeats and confers a broad spectrum of blast resistance. We then analyzed the sequences of the Ptr gene from weedy rice, Ptr^BHA, and identified a unique amino acid glutamine at protein position 874. Minor changes of protein conformation of the Ptr^BHAgene were predicted through structural analysis of Ptr^BHA, suggesting that the product of Ptr^BHAis involved in disease resistance. A gene-specific codominant marker HJ17-13 from Ptr^BHAwas then developed to distinguish alleles in weeds and crops. The Ptr^BHAgene existed in 207 individuals of the same mapping population, where qBR12.3b was mapped using this gene-specific marker. Disease reactions of 207 individuals and their parents to IB-33 were evaluated. The resistant individuals had Ptr^BHAwhereas the susceptible individuals did not, suggesting that HJ17-13 is reliable to predict qBR12.3b. Taken together, this newly developed marker, and weedy rice genotypes carrying qBR12.3b, are useful for blast improvement using marker assisted selection.