Rajeev Rathour

Analysis of Magnaporthe oryzae Genome Reveals a Fungal Effector, Which Is Able to Induce Resistance Response in Transgenic Rice Line Containing Resistance Gene, Pi54

Rice blast caused by Magnaporthe oryzae is one of the most important diseases of rice. Pi54, a rice gene that imparts resistance to M. oryzae isolates prevalent in India, was already cloned but its avirulent counterpart in the pathogen was not known. After decoding the whole genome of an avirulent isolate of M. oryzae, we predicted 11440 protein coding genes and then identified four candidate effector proteins which are exclusively expressed in the infectious structure, appresoria. In silico protein modeling followed by interaction analysis between Pi54 protein model and selected four candidate effector proteins models revealed that Mo-01947_9 protein model encoded by a gene located at chromosome 4 of M. oryzae, interacted best at the Leucine Rich Repeat domain of Pi54 protein model. Yeast-two-hybrid analysis showed that Mo-01947_9 protein physically interacts with Pi54 protein. Nicotiana benthamiana leaf infiltration assay confirmed induction of hypersensitive response in the presence of Pi54 gene in a heterologous system. Genetic complementation test also proved that Mo-01947_9 protein induces avirulence response in the pathogen in presence of Pi54 gene. Here, we report identification and cloning of a new fungal effector gene which interacts with blast resistance gene Pi54 in rice.

Extensive sequence variation in rice blast resistance gene Pi54 makes it broad spectrum in nature.

Rice blast resistant gene, Pi54 cloned from rice line, Tetep, is effective against diverse isolates of Magnaporthe oryzae. In this study, we prospected the allelic variants of the dominant blast resistance gene from a set of 92 rice lines to determine the nucleotide diversity, pattern of its molecular evolution, phylogenetic relationships and evolutionary dynamics, and to develop allele specific markers. High quality sequences were generated for homologs of Pi54 gene. Using comparative sequence analysis, InDels of variable sizes in all the alleles were observed. Profiling of the selected sites of SNP (Single Nucleotide Polymorphism) and amino acids (N sites ≥ 10) exhibited constant frequency distribution of mutational and substitutional sites between the resistance and susceptible rice lines, respectively. A total of 50 new haplotypes based on the nucleotide polymorphism was also identified. A unique haplotype (H_3) was found to be linked to all the resistant alleles isolated from indica rice lines. Unique leucine zipper and tyrosine sulfation sites were identified in the predicted Pi54 proteins. Selection signals were observed in entire coding sequence of resistance alleles, as compared to LRR domains for susceptible alleles. This is a maiden report of extensive variability of Pi54 alleles in different landraces and cultivated varieties, possibly, attributing broad-spectrum resistance to Magnaporthe oryzae. The sequence variation in two consensus region: 163 and 144 bp were used for the development of allele specific DNA markers. Validated markers can be used for the selection and identification of better allele(s) and their introgression in commercial rice cultivars employing marker assisted selection.

Marker assisted pyramiding of major blast resistance genes Pi9 and Pita in the genetic background of an elite Basmati rice variety, Pusa Basmati 1

Basmati is a premium quality rice of India which is highly priced in the international market. Pusa Basmati 1, an elite Basmati rice variety is highly susceptible to rice blast caused by Magnaporthe oryzae. Therefore, pyramiding blast resistance genes is essential to effectively combat the blast disease and increase the durability of resistance genes. The blast resistance genes Pi9 and Pita have been earlier demonstrated to be effective in Basmati growing regions of the country. Therefore, in the present study, monogenicnear isogenic lines Pusa 1637-18-7-6-20 and Pusa 1633-3-88-16-1 carrying Pi9 and Pita, respectively, were intercrossed to generate pyramided lines through marker assisted foreground, background and phenotypic selection for recurrent parent phenotype. The pyramided lines carrying Pi9+Pita were found to be either at par or superior to the recurrent parent Pusa Basmati 1 for agro-morphological, grain and cooking quality traits. Further, these pyramided lines were also found to show resistance against three virulent pathotypes of M. oryzae namely, Mo-nwi-kash 1, Mo-nwi-lon2 and Mo-ei-ran1, when evaluated under artificial inoculation conditions as well as in the natural epiphytotic conditions of uniform blast nursery at two locations. The developed pyramided lines are the potential sources of blast resistance genes in the Basmati improvement program and can also be released for commercial cultivation after required testing.

Phenotyping of improved rice lines and landraces for blast and sheath blight resistance

Globally, two major fungal diseases of rice,viz., blast caused by Magnaporthe oryzae and sheath blight incited by Rhizoctonia solani Kuhn. occur in all agro-ecosystems resulting in significant yield losses. In the present study, a set of 100 rice lines were evaluated for blast and sheath blight resistance. Twenty seven genotypes were identified to be resistant against M. oryzae isolate (Mo-ni-0066). However, evaluation of these genotypes with highly virulent M. oryzae Karnataka isolate (Mo-si-mnd) revealed that only two genotypes viz., Bhusan San and P1490-03 were resistant. Same set of genotypes were screened for sheath blight resistance. Four entries, viz., BPL7-12, BML27-1, BML 21-1 and Kajarahwa possess high degree of tolerance to sheath blight but none of the rice entries was completely resistant.

Identification and validation of rice blast resistance genes in Indian rice germplasm

Blast disease caused by Magnaporthe oryzae is a major constraint in rice production. Identification of new donors for blast resistance is a pre-requisite for effective utilization of diverse germplasm for marker assisted incorporation of blast resistance into improved varieties. Therefore, in the present study, a set of 100 diverse rice germplasm accessions were evaluated for 11 blast resistance genes namely Pikm, Pik, Pikh, Pi1, Pi5, Pi54, Pib, Piz5, Piz, Pi9 and Pish, both at genotypic and phenotypic level. Genotyping with gene based/gene linked markers could identify six genotypes from the germplasm possessing as many as six resistance specific alleles. A total of 34 and 67 germplasm lines were found to possess resistance alleles for two genes, Pikm and Pik, respectively. Phenotypic validation using artificial inoculation in the germplasm was carried out with 4 diverse isolates under controlled conditions. The congruence between marker genotype and disease phenotype on a set of monogenic lines for blast resistance in the LTH background was used to compute Disease Resistance Index (DRI) in the germplasm. Cumulative DRI for each genotype was computed over all the marker loci. The genotypes Heibao, Kalinga-I, Vijetha, Anjali, Bhaubhog, Sada Kaijam, Kala Jeera had high cumulative resistance score. Allelic Cumulative Disease Resistance Index (ACDRI), a measure for comparing the effectiveness of markers was calculated and markers linked to Pikm, Pik, Piz5, Pi1 were found to possess higher accuracy and better correlation with expected patterns of resistance under artificial inoculation. Based on disease resistance index, 25 germplasm accessions were found carrying blast resistance specific alleles at different loci and were fully validated for disease phenotype, which are valuable in breeding for resistance, allele mining and functional genomics studies.

Co-evolutionary interactions between host resistance and pathogen avirulence genes in rice-Magnaporthe oryzae pathosystem

Rice and Magnaporthe oryzae constitutes an ideal pathosystem for studying host-pathogen interaction in cereals crops. There are two alternative hypotheses, viz. Arms race and Trench warfare, which explain the co-evolutionary dynamics of hosts and pathogens which are under continuous confrontation. Arms race proposes that both R- and Avr- genes of host and pathogen, respectively, undergo positive selection. Alternatively, trench warfare suggests that either R- or Avr- gene in the pathosystem is under balanced selection intending to stabilize the genetic advantage gained over the opposition. Here, we made an attempt to test the above-stated hypotheses in rice-M. oryzae pathosystem at loci of three R-Avr gene pairs, Piz-t-AvrPiz-t, Pi54-AvrPi54 and Pita-AvrPita using allele mining approach. Allele mining is an efficient way to capture allelic variants existing in the population and to study the selective forces imposed on the variants during evolution. Results of nucleotide diversity, neutrality statistics and phylogenetic analyses reveal that Piz-t, Pi54 and AvrPita are diversified and under positive selection at their corresponding loci, while their counterparts, AvrPiz-t, AvrPi54 and Pita are conserved and under balancing selection, in nature. These results imply that rice-M. oryzae populations are engaged in a trench warfare at least at the three R/Avr loci studied. It is a maiden attempt to study the co-evolution of three R-Avr gene pairs in this pathosystem. Knowledge gained from this study will help in understanding the evolutionary dynamics of host-pathogen interaction in a better way and will also aid in developing new durable blast resistant rice varieties in future.

Development and validation of co-dominant gene based markers for Pi9, a gene governing broad-spectrum resistance against blast disease in rice

Rice production and grain quality are severely affected by blast disease caused by the ascomycetous fungus Magnaporthe oryzae. Incorporation of genes that confer broad-spectrum resistance to blast has been a priority area in rice breeding programs. The blast resistance gene Pi9 sourced from Oryza minuta has shown broad spectrum and durable resistance to blast world-wide. In the present study co-dominant gene-based markers were developed for the precise marker-assisted tracking of Pi9 in breeding programs. The developed markers were validated across a diverse set of cultivars including basmati, indica and japonica varieties. Two markers, Pi9STS-1 and Pi9STS-2, effectively differentiated Pi9 donors from all the indicas and commercial basmati varieties tested. However, these markers were monomorphic between Pi-9 donors (IRBL9-W and Pusa 1637) and japonica type varieties. An additional gene-derived CAPS marker Pi91F_ 2R was developed to differentiate Pi9 donors from japonicas and traditional basmati lines. The co-dominant markers developed in the present study will be of immense utility to rice breeders for precise and speedy incorporation of Pi-9 into susceptible rice varieties through marker-assisted selection.