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.

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.

HETEROZYGOUS ORYZA SATIVA L. DOUBLED HAPLOID GENERATED THROUGH IN VITRO ANDROGENESIS

In the course of marker-assessted selection for the blast resistance gene Pi-b, among 372 rice Oryza sativa L. doubled haploids DH0 geterozygous plant was identified. In the offspring (11 doubled haploids DH1), only Pi-b resistance allele was revealed. The reason for the appearance of false heterozygosity is discussed. By analogy with mixoploidy, an explanation is proposed for the phenomenon of somatic tissues mixogeny.

Effectiveness of the Wheat Blast Resistance Gene Rmg8 in Bangladesh Suggested by Distribution of an AVR-Rmg8 Allele in the Pyricularia oryzae Population

Wheat blast caused by the Triticum pathotype of Pyricularia oryzae was first reported in 1985 in Brazil and recently spread to Bangladesh. We tested whether Rmg8 and RmgGR119, recently identified resistance genes, were effective against Bangladeshi isolates of the pathogen. Common wheat accessions carrying Rmg8 alone (IL191) or both Rmg8 and RmgGR119 (GR119) were inoculated with Brazilian isolates (Br48, Br5, and Br116.5) and Bangladeshi isolates (T-108 and T-109). Br48, T-108, and T-109 carried the eI type of AVR-Rmg8 (the avirulence gene corresponding to Rmg8) while Br5 and Br116.5 carried its variants, eII and eII’ types, respectively. Detached primary leaves of IL191 and GR119 were resistant to all isolates at 25°C. At a higher temperature (28°C), their resistance was still effective against the eI carriers but was reduced to a low level against the eII/eII’ carriers. A survey of databases and sequence analyses revealed that all Bangladeshi isolates carried the eI type which induced a higher level of resistance than the eII/eII’ types. The resistance of IL191 (Rmg8/−) to the eI carriers was maintained even at the heading stage and at the higher temperature. In addition, GR119 (Rmg8/RmgGR119) displayed higher levels of resistance than IL191 at this stage. These results suggest that Rmg8 combined with RmgGR119 will be useful in breeding for resistance against wheat blast in Bangladesh.

Molecular analysis of five rice blast resistant genes in landraces from Myanmar and Laos

In order to understand rice blast resistance gene patterns in rice landraces from Myanmar and Laos, we analysed the Pita, Pib, Pikh, Pi9 and Pi5 genes in 80 landraces through the use of functional molecular markers and functional fragment sequencing. These landraces were separated into four haplotypes (H1–H4) based upon three mutations identified in the Pita sequence. All four haplotypes were detected among landraces from Myanmar, whereas only the H1 and H2 haplotypes were detected among landraces from Laos. We additionally grouped landraces carrying 0–4 resistance genes into 18 genotypes based upon their resistance-susceptibility patterns and found that 16 of these genotypes were detected among Myanmar landraces at relative frequencies ranging from 2.50–12.50%, while 10 were detected among landraces from Laos at relative frequencies of 2.50–42.50%. The ‘Pib( + ) pikh(−) pi9(−) pi5(−) pita(−)’ genotype was found to be dominant, accounting for 12.5 and 42.5% of landraces from Myanmar and Laos, respectively. The Pib, Pikh, Pi5 and Pita genes were detected in 52.50, 32.50, 37.50 and 30.00% of landraces from Myanmar respectively, whereas they were detected in 52.50, 12.50, 30.00 and 17.50% of landraces from Laos, respectively. The Pi9 gene was rarely detected among analysed landraces and was particularly rare in those from Laos. These findings suggest that rice landraces from Myanmar are more genetically diverse than those from Laos, with clear differences in blast resistance gene patterns between landraces from these two countries.