Effector genes in Magnaporthe oryzae Triticum as Potential Targets for Incorporating Blast Resistance in Wheat

Wheat blast (WB), caused by Magnaporthe oryzae Triticum pathotype, recently emerged as a destructive disease that threatens global wheat production. Since few sources of genetic resistance have been identified in wheat, genetic transformation of wheat with rice blast resistance genes could expand resistance to WB. We evaluated the presence/absence of homologs of rice blast effector genes in Triticum isolates with the aim of identifying avirulence genes in field populations whose cognate rice resistance genes could potentially confer resistance to WB. We also assessed presence of the wheat pathogen AVR-Rmg8 gene, and identified new alleles. A total of 102 isolates collected in Brazil, Bolivia and Paraguay from 1986 to 2018 were evaluated by PCR using 21 pairs of gene-specific primers. Effector gene composition was highly variable, with homologs to AvrPiz-t, AVR-Pi9, AVR-Pi54 and ACE1 showing the highest amplification frequencies (>94%). We identified Triticum isolates with a functional AvrPiz-t homolog that triggers Piz-t-mediated resistance in the rice pathosystem, and produced transgenic wheat plants expressing the rice Piz-t gene. Seedlings and heads of the transgenic lines were challenged with isolate T25 carrying functional AvrPiz-t. Although slight decreases in the percentage of diseased spikelets and leaf area infected were observed in two transgenic lines, our results indicated that Piz-t did not confer useful WB resistance. Monitoring of avirulence genes in populations is fundamental to identifying effective resistance genes for incorporation into wheat by conventional breeding or transgenesis. Based on avirulence gene distributions, rice resistance genes Pi9 and Pi54 might be candidates for future studies.

Superior japonica rice variety YJ144 with improved rice blast resistance, yield, and quality achieved using molecular design and multiple breeding strategies

Yanfeng 47 (YF47) is an elite japonica rice variety cultivated in China on nearly 2 million hectares over the past 20 years. However, YF47 is highly susceptible to rice blast (Magnaporthe oryzae), one of the most destructive rice diseases. In this study, we developed novel TPAP (tetra-primer ARMS-PCR) functional markers for the genes Pita, Pib and Pid2, all of which afford broad-spectrum resistance to blast. A collection of 91 japonica rice germplasms with similar ecological characteristics to YF47 was screened, and Wuyunjing 27 (WYJ27) with Pita and Pib alleles, and P135 with the Pid2 allele, were identified. Furthermore, the corresponding positive Pita, Pib and Pid2 alleles were transferred into YF47 using single, mutual and backcrosses, together with molecular marker-assisted selection (MAS) and anther culture technology. These genetic materials, carrying one, two, or three functional alleles, were generated within three years, and compared to YF47 they all showed improved resistance to naturally inoculated rice blast. Further improved lines (IL) 1 to 5 (all containing Pita, Pib and Pid2 alleles) were evaluated for yield performance, and when no fungicide was applied all lines except IL-4 showed increased traits compared with those of YF47. IL-5, renamed Yanjing 144 (YJ144), showed yield increases in the Liaoning province regional variety comparison test and superior appearance quality compared to YF47. Our work provides a molecular design strategy for pyramiding multiple beneficial genes to rapidly improve rice blast resistance, yield, and quality using multiple breeding strategies.

Association and Transcription Analyses Identify Candidate Susceptibility Genes for Rice Blast Resistance Breeding

Traditional rice blast resistance breeding largely depends on utilizing the typical resistance (R) genes. The lack of durable R genes prompts rice breeders to find new source of resistance. The susceptibility (S) genes could be the potentially useful targets for genetic engineering using the cutting-edging genome-editing technologies. However, how to identify S genes is challenging. Here, by combining genome-wide association study and transcriptional analysis, we identified the candidate genes in the rice genome whose expression levels were negatively associated with rice blast resistance. We initially identified five candidate genes by qRT-PCR and comparison of their sequences among the rice blast resistant and susceptible varieties. The obtained results showed that the polymorphisms in 3’ un-translated regions (3’-UTR) of two genes, Os02g39360 and Os08g29170, named RNG1 and RNG3, respectively, were tightly associated with their expression levels and rice blast susceptibility. Subsequently, the negatively association between the expression levels and blast resistance was confirmed using the 3K rice population. Meanwhile, the frequency of the resistance genotype of these two genes was significantly decreased during the first step of rice domestication from wild rice to landrace rice. In contrast, the frequency was increased during the second step of domestication from landrace rice to modern cultivars. Nucleotide diversity analysis found an obvious selective sweep in the surrounding loci of RNG3, means that RNG3 has been artificially selected. The results obtained in this study provide new targets for further gene functional analysis as well as genome editing-based rice blast resistance breeding.

Cloning and Function Analysis of a Novel Rice Blast Resistance Gene Pi65 in Japonica Rice

Rice blast seriously threatens rice production worldwide. Utilizing the rice blast resistance gene to breed the rice blast resistant varieties is one of the best ways to control rice blast disease. Using a map-based cloning strategy, here, we cloned a novel rice blast resistance gene, Pi65 from the resistant variety GangYu129 (abbreviated GY129, O. sativa japonica ). Overexpression of Pi65 in the susceptible variety LiaoXing1 (abbreviated LX1, O. sativa japonica ) enhanced rice blast resistance, while knockout of Pi65 in GY129 resulted in susceptible to rice blast disease. Pi65 encodes two transmembrane domains, with 15 LRR domains and one serine/threonine protein kinase catalytic domain, conferring resistance to isolates of M. oryzae collected from northeast China. There are sixteen amino acids differences between the Pi65 resistance and susceptible alleles. Compared with the Pi65 resistant allele, the susceptible allele deleted one LRR domain. Pi65 was constitutively expressed in whole plants, and it could be induce expressed in the early stage of M. oryzae infection . Transcriptome analysis revealed that numerous genes associated with disease resistance were specifically upregulated in GY129 24-hour post inoculation (HPI), on the contrary, the photosynthesis-and carbohydrate metabolism-related genes were particularly downregulated 24 HPI, demonstrating that the disease resistance associated genes has been activated in GY129 (carrying Pi65 ) after rice blast fungal infection, and the cellular basal and energy metabolism was inhibited simultaneously. Our study provides genetic resources for improving rice blast resistance as well as enriches the study of rice blast resistance mechanisms.

An Efficient Genetic Manipulation Protocol for Dark Septate Endophyte Falciphora Oryzae

Falciphora oryzae is a dark septate endophyte (DSE) isolated from wild rice roots (Oryza sativa L.). It was classified as a non-clavicitaceous endophyte. The fungus colonizes rice roots, showing a significant increase in agronomic parameters with plant biomass, rice blast resistance, yield, and quality. The construction of the genetic manipulation system is critical to study the relationship between F. oryzae and O. sativa. In the present study, the protoplast preparation and transformation system of F. oryzae was investigated. The key parameters affecting the efficiency of protoplast production, such as osmotic pressure stabilizer, enzyme digestion conditions, and fungal age, were studied. The results showed that F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9mol L-1 KCl solution and 10 mg mL−1 glucanase at 30℃ with shaking 80 rpm for 2-3 h. When the protoplasts were plated on a regenerations-agar (RgA) medium containing 1M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired GFP-expressing transformants by transforming the pKD6-GFP vector into protoplasts. Further, the GFP expression in fungal hyphae possessed good stability and intensity during symbiosis in rice roots.The genetic manipulation system of endophytic fungus facilitates the further exploration the interaction between the endophytic fungus and their hosts.

A Blast Resistance Gene Pi65 with LRR-RLK Domain is Required for Resistance to M. Oryzae

Rice blast seriously threatens rice production worldwide. To control this disease, it is necessary to identify and utilize blast resistance genes to breed disease-resistant rice varieties. Here, we report a rice blast resistance gene, Pi65, isolated from the resistant variety GangYu129 (abbreviated GY129, O. sativa japonica) by map-based cloning. Pi65 overexpression in the susceptible variety LiaoXing1 (abbreviated LX1, O. sativa japonica) enhanced blast resistance, while Pi65 knockout in GY129 resulted in a decrease in its resistance to rice blast. Pi65 encodes two transmembrane regions, with 15 LRR domains and one serine/threonine protein kinase catalytic domain, conferring resistance to isolates of M. oryzae collected from northeast China. Sixteen amino acids differed between the resistance and susceptibility proteins. The Pi65 susceptibility allele had one fewer LRR duplication. Pi65 was constitutively expressed in whole plants, and M. oryzae inoculation significantly increased its expression level. Transcriptome sequencing revealed that numerous genes associated with disease resistance were specifically upregulated in GY129 24 h after M. oryzae inoculation, and photosynthesis-and carbohydrate metabolism-related genes were particularly downregulated, demonstrating disease resistance gene activation in GY129 mediated by Pi65 after rice blast fungal infection, cellular basal and energy metabolism was inhibited simultaneously. These combined factors endow GY129 with rice blast resistance. Our study provides genetic resources for improving rice blast resistance in japonica rice and enriches the study of rice blast resistance mechanisms.

The Rice Blast Resistance Gene Pid Family has been Strictly Diverged into Indica and Japonica Subspecies

Background: Rice blast (causative agent the fungus Pyricularia oryzae) represents a major constraint over the productivity of one of the world’s most important staple foods. Genes encoding resistance have been identified in both the indica and japonica subspecies genepools, and combining these within new cultivars represents a rational means of combating the pathogen.Results: In this research, a deeper allele mining was carried out on Pid-2, Pid-3, and Pid-4 by their specific FNP markers in the three panels consisting of 70 indica and 58 japonica cultivars. Within Pid-2, three functional and one non-functional alleles were identified; the former were only identified in indica type entries. At Pid-3, four functional and one non-functional alleles were identified, and once again, all of the former were present in indica type entries. However, the pattern of variation at Pid-4 was rather different: here, the five functional alleles uncovered were dispersed across the japonica type germplasm. Among all the 12 candidate functional alleles, both Pid2-ZS and Pid3-ZS were predominant.Conclusions: Variation in both Pid-2 and Pid-3 appears to have evolved in response to pathogen pressure exerted on indica type cultivars, while that in Pid-4 reflects the interaction between the fungus and the host in japonica type crops. Owning to the founder lineage, ‘Zhenzhuai 11-ZS97’, rather limited genotypes of the Pid family have been effective in both indica and japonica rice groups, of which Pid2-ZS+Pid3-ZS is present in a large proportion of Chinese indica cultivars released since the 1960s.