Functional complementation of rice blast resistance gene Pi-k h (Pi54) conferring resistance to diverse strains of Magnaporthe oryzae

Blast disease of rice, caused by Magnaporthe oryzae is an explosive disease that can spread rapidly in conducive conditions. R-gene mediated resistance offers an environmentally sustainable solution for management of this important disease of rice. We have earlier identified a unique R-gene of rice, on chromosome 11 of Oryza sativa ssp. indica cultivar Tetep. In this study we report functional validation of the Pi-k ^h (Pi54) gene using complementation assay. The blast resistance candidate gene Pi-k ^h (Pi54) was cloned into a plant transformation vector and the construct was used to transform a japonica cultivar of rice Taipei 309, which is susceptible to M. oryzae. Transgenic lines containing Pi-k ^h (Pi54) gene were found to confer high degree of resistance to diverse isolates of M. oryzae. The callose deposition was analyzed and compared between the transgenic and non-transgenic rice plants and widespread deposition was observed at the infection sites in plants showing incompatible interaction. Successful complementation of Pi-k ^h (Pi54) gene confirmed that the gene is responsible for resistance to M. oryzae in transgenic lines developed during this study. Expression analysis of the gene in resistant plants revealed that the gene is pathogen inducible in nature and is not expressed constitutively. Detection of callose deposition in resistant plants containing Pi-k ^h (Pi54) gene implicates its involvement in the initiation of defense response cascade.     

The isolation of Pi1, an allele at the Pik locus which confers broad spectrum resistance to rice blast

We report the isolation of Pi1, a gene conferring broad-spectrum resistance to rice blast (Magnaporthe oryzae). Using loss- and gain-of-function approaches, we demonstrate that Pi1 is an allele at the Pik locus. Like other alleles at this locus, Pi1 consists of two genes. A functional nucleotide polymorphism (FNP) was identified that allows differentiation of Pi1 from other Pik alleles and other non-Pik genes. A extensive germplasm survey using this FNP reveals that Pi1 is a rare allele in germplasm collections and one that has conferred durable resistance to a broad spectrum of pathogen isolates.     

A Pid3 allele from rice cultivar Gumei2 confers resistance to Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases. Using map-based strategy and in silico approach we isolated a new rice (Oryza sativa L.) blast resistance allele of Pid3, designated Pi25, from a stable blast resistance cultivar Gumei2. Overexpression analysis and complementation test showed that Pi25 conferred blast resistance to M. oryzae isolate js001-20. Sequence analysis showed that Pi25 was an intronless gene of 2772 nucleotides with single nucleotide substitution in comparison to Pid3 at the nucleotide position 459 and predicatively encoded a typical coiled coil-nucleotide binding site-leucine rich repeat (CC--NBS--LRR) protein of 924 amino acid residuals with 100% identity to Pid3 putative protein. The susceptible allele pi25 in Nipponbare contained a nonsense mutation at the nucleotide position 2209 resulting in a truncated protein with 736 amino acid residuals. In addition, 14 nucleotide substitutions resulting in 10 amino acid substitutions were identified between Pi25 and pi25 upstream the premature stop codon in the susceptible allele. Although the mechanism of Pi25/Pid3-mediated resistance needs to be further investigated, the isolation of the allele would facilitate the utilization of Pi25/Pid3 in rice blast resistance breeding program via transgenic approach and marker assisted selection.     

Diversity of centromeric repeats in two closely related wild rice species, Oryza officinalis and Oryza rhizomatis

Oryza officinalis (CC, 2n=24) and Oryza rhizomatis (CC, 2n=24) belong to the Oryza genus, which contains more than 20 identified wild rice species. Although much has been known about the molecular composition and organization of centromeres in Oryza sativa, relatively little is known of its wild relatives. In the present study, we isolated and characterized a 126-bp centromeric satellite (CentO-C) from three bacterial artificial chromosomes of O. officinalis. In addition to CentO-C, low abundance of CentO satellites is also present in O. officinalis. In order to determine the chromosomal locations and distributions of CentO-C (126-bp), CentO (155 bp) and TrsC (366 bp) satellite within O. officinalis, fluorescence in situ hybridization examination was done on pachytene or metaphase I chromosomes. We found that only ten centromeres (excluding centromere 7 and 2) contain CentO-C arrays in O. officinalis, while centromere 7 comprises CentO satellites, and centromere 2 is devoid of any detectable satellites. For TrsC satellites, it was detected at multiple subtelomeric regions in O. officinalis, however, in O. rhizomatis, TrsC sequences were detected both in the four centromeric regions (CEN 3, 4, 10, 11) and the multiple subtelomeric regions. Therefore, these data reveal the evolutionary diversification pattern of centromere DNA within/or between close related species, and could provide an insight into the dynamic evolutionary processes of rice centromere.     

Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta to cultivated rice,Oryza sativa

Summary Oryza minuta J. S. Presl ex C. B. Presl is a tetraploid wild rice with resistance to several insects and diseases, including blast (caused by Pyricularia grisea) and bacterial blight (caused by Xanthomonas oryzae pv. oryzae). To transfer resistance from the wild species into the genome of cultivated rice (Oryza sativa L.), backcross progeny (BC₁, BC₂, and BC₃) were produced from interspecific hybrids of O. sativa cv IR31917-45-3-2 (2n=24, AA genome) and O. minuta Acc. 101141 (2n=48, BBCC genomes) by backcrossing to the O. sativa parent followed by embryo rescue. The chromosome numbers ranged from 44 to 47 in the BC₁ progeny and from 24 to 37 in the BC₂ progeny. All F₁ hybrids were resistant to both blast and bacterial blight. One BC₁ plant was moderately susceptible to blast while the rest were resistant. Thirteen of the 16 BC₂ progeny tested were resistant to blast; 1 blast-resistant BC₂, plant 75-1, had 24 chromosomes. A 3 resistant: 1 susceptible segregation ratio, consistent with the action of a major, dominant gene, was observed in the BC₂F₂ and BC₂F₃ generations. Five of the BC₁ plants tested were resistant to bacterial blight. Ten of the 21 BC₂ progeny tested were resistant to Philippine races 2, 3, and 6 of the bacterial blight pathogen. One resistant BC₂, plant 78-1, had 24 chromosomes. The segregation of reactions of the BC₂F₂, BC₂F₃, and BC₂F₄ progenies of plant 78-1 suggested that the same or closely linked gene(s) conferred resistance to races 2, 3, 5, and 6 of the bacterial blight pathogen from the Philippines.     

A novel gene, <Emphasis Type="Italic">Pi40(t)</Emphasis>, linked to the DNA markers derived from NBS-LRR motifs confers broad spectrum of blast resistance in rice

Rice blast disease caused by Magnaporthe grisea is a continuous threat to stable rice production worldwide. In a modernized agricultural system, the development of varieties with broad-spectrum and durable resistance to blast disease is essential for increased rice production and sustainability. In this study, a new gene is identified in the introgression line IR65482-4-136-2-2 that has inherited the resistance gene from an EE genome wild Oryza species, O. australiensis (Acc. 100882). Genetic and molecular analysis localized a major resistance gene, Pi40(t), on the short arm of chromosome 6, where four blast resistance genes (Piz, Piz-5, Piz-t, and Pi9) were also identified, flanked by the markers S2539 and RM3330. Through e-Landing, 14 BAC/PAC clones within the 1.81-Mb equivalent virtual contig were identified on Rice Pseudomolecule3. Highly stringent primer sets designed for 6 NBS-LRR motifs located within PAC clone P0649C11 facilitated high-resolution mapping of the new resistance gene, Pi40(t). Following association analysis and detailed haplotyping approaches, a DNA marker, 9871.T7E2b, was identified to be linked to the Pi40(t) gene at the 70 Kb chromosomal region, and differentiated the Pi40(t) gene from the LTH monogenic differential lines possessing genes Piz, Piz-5, Piz-t, and Pi-9. Pi40(t) was validated using the most virulent isolates of Korea as well as the Philippines, suggesting a broad spectrum for the resistance gene. Marker-assisted selection (MAS) and pathotyping of BC progenies having two japonica cultivar genetic backgrounds further supported the potential of the resistance gene in rice breeding. Our study based on new gene identification strategies provides insight into novel genetic resources for blast resistance as well as future studies on cloning and functional analysis of a blast resistance gene useful for rice improvement.     

Expression of Dm-AMP1 in rice confers resistance to Magnaporthe oryzae and Rhizoctonia solani

Magnaporthe oryzae and Rhizoctonia solani, are among the most important pathogens of rice, severely limiting its productivity. Dm-AMP1, an antifungal plant defensin from Dahlia merckii, was expressed in rice (Oryza sativa L. sp. indica cv. Pusa basmati 1) using Agrobacterium tumefaciens-mediated transformation. Expression levels of Dm-AMP1 ranged from 0.43% to 0.57% of total soluble protein in transgenic plants. It was observed that constitutive expression of Dm-AMP1 suppresses the growth of M. oryzae and R. solani by 84% and 72%, respectively. Transgenic expression of Dm-AMP1 was not accompanied by an induction of pathogenesis-related (PR) gene expression, indicating that the expression of DmAMP1 directly inhibits the pathogen. The results of in vitro, in planta and microscopic analyses suggest that Dm-AMP1 expression has the potential to provide broad-spectrum disease resistance in rice.     

Improving blast resistance of maintainer line DRR 9B by transferring broad spectrum resistance gene Pi2 by marker assisted selection in rice

Hybrid rice technology offers great promise to further enhance rice production and productivity for global food security. Improving hybrid rice parental lines is the first step in developing heterotic rice hybrids. To improve resistance against blast disease, a maintainer line DRR 9B was fortified with a major broad-spectrum blast resistance gene Pi2 through marker-assisted selection. The rice blast caused by Magnaporthe oryzae is a major disease and can cause severe yield losses upto 100%. The NILs of Samba Mahsuri namely BA-23-11-89-12-168 possessing Pi2 was utilized as a donor parent. The PCR-based molecular marker tightly linked to Pi2 gene was used for the foreground selection at BC₁F₁ generation. The molecular marker tightly linked to the major fertility restorer gene Rf4 was used for negative selection (i.e., selection of plants possessing non fertility restoring alleles) at BC₁F₁ generation to identify maintainer lines. The positive plants with Rf4 gene were added to the restorer pool for restorer line development. At each stage, MAS for Pi2 coupled with stringent phenotypic selection for agro-morphological and grain quality traits were exercised. At BC₁F₃ generation, one hundred families were screened against blast disease at uniform blast nursery (UBN) and selected resistant lines were advanced to next generations. In the BC₁F₅ generation plants were subjected to agro-morphological evaluation for yield and yield-contributing traits. The selected plants at BC₁F₅ generation were crossed with DRR 9A to assess the maintainer ability of blast resistance lines and for further CMS line conversion for hybrid rice breeding for developing blast resistance rice hybrids.     

Sequence variation of avirulence gene AVR-Pita1 in rice blast fungus, Magnaporthe oryzae

The interaction between rice, Oryza sativa, and rice blast fungus, Magnaporthe oryzae, is triggered by an interaction between the protein products of the host resistant gene, and the pathogen avirulence gene. This interaction follows the ‘gene-for-gene' concept. The resistant gene has effectively protected rice plants from rice blast infection. However, the resistant genes usually break down several years after the release of the resistant rice varieties because the fungus has evolved to new races. The objective of this study is to investigate the nucleotide sequence variation of the AVR-Pita1 gene that influences the adaption of rice blast fungus to overcome the resistant gene, Pi-ta. Thirty rice blast fungus isolates were collected in 2005 and 2010 from infected rice plants in northern and northeastern Thailand. The nucleotide sequences of AVR-Pita1 were amplified and analyzed. Phylogenetic analysis was conducted using the MEGA 5.0 program. The results showed a high level of nucleotide sequence polymorphisms and the positive genetic selection pressure in Thai rice blast isolates. The details of sequence variation analysis were described in this article. The information from this study can be used for rice blast resistant breeding program in the future.     

Perception of the chitin oligosaccharides contributes to disease resistance to blast fungus Magnaporthe oryzae in rice

Chitin is a component of fungal cell walls, and its fragments act as elicitors in many plants. The plasma membrane glycoprotein CEBiP, which possesses LysM domains, is a receptor for the chitin elicitor (CE) in rice. Here, we report that the perception of CE by CEBiP contributes to disease resistance against the rice blast fungus, Magnaporthe oryzae, and that enhanced responses to CE by engineering CEBiP increase disease tolerance. Knockdown of CEBiP expression allowed increased spread of the infection hyphae. To enhance defense responses to CE, we constructed chimeric genes composed of CEBiP and Xa21, which mediate resistance to rice bacterial leaf blight. The expression of either CRXa1 or CRXa3, each of which contains the whole extracellular portion of CEBiP, the whole intracellular domain of XA21, and the transmembrane domain from either CEBiP or XA21, induced cell death accompanied by an increased production of reactive oxygen and nitrogen species after treatment with CE. Rice plants expressing the chimeric receptor exhibited necrotic lesions in response to CE and became more resistant to M. oryzae. Deletion of the first LysM domain in CRXA1 abolished these cellular responses. These results suggest that CEs are produced and recognized through the LysM domain of CEBiP during the interaction between rice and M. oryzae and imply that engineering pattern recognition receptors represents a new strategy for crop protection against fungal diseases.     

Genome‐wide association study identifies an NLR gene that confers partial resistance to Magnaporthe oryzae in rice

Because of the frequent breakdown of major resistance (R) genes, identification of new partial R genes against rice blast disease is an important goal of rice breeding. In this study, we used a core collection of the Rice Diversity Panel II (C‐RDP‐II), which contains 584 rice accessions and are genotyped with 700 000 single‐nucleotide polymorphism (SNP) markers. The C‐RDP‐II accessions were inoculated with three blast strains collected from different rice‐growing regions in China. Genome‐wide association study identified 27 loci associated with rice blast resistance (LABRs). Among them, 22 LABRs were not associated with any known blast R genes or QTLs. Interestingly, a nucleotide‐binding site leucine‐rich repeat (NLR) gene cluster exists in the LABR12 region on chromosome 4. One of the NLR genes is highly conserved in multiple partially resistant rice cultivars, and its expression is significantly up‐regulated at the early stages of rice blast infection. Knockout of this gene via CRISPR‐Cas9 in transgenic plants partially reduced blast resistance to four blast strains. The identification of this new non‐strain specific partial R gene, tentatively named rice blast Partial Resistance gene 1 (PiPR1), provides genetic material that will be useful for understanding the partial resistance mechanism and for breeding durably resistant cultivars against blast disease of rice.     

Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus ( Magnaporthe grisea) in transgenic rice

Transgenic rice ( Oryza sativa cv. Sasanishiki) overexpressing the wasabi defensin gene, a plant defensin effective against the rice blast fungus, was generated by Agrobacterium tumefaciens-mediated transformation. Twenty-two T2 homozygous lines harboring the wasabi defensin gene were challenged by the blast fungus. Transformants exhibited resistance to rice blast at various levels. The inheritance of the resistance over generations was investigated. T3 plants derived from two highly blast-resistant T2 lines (WT14-5 and WT43-5) were challenged with the blast fungus using the press-injured spots method. The average size of disease lesions of the transgenic line WT43-5 was reduced to about half of that of non-transgenic plants. The 5-kDa peptide, corresponding to the processed form of the wasabi defensin, was detected in the total protein fraction extracted from the T3 progeny. Transgenic rice plants overproducing wasabi defensin are expected to possess a durable and wide-spectrum resistance (i.e. field resistance) against various rice blast races.     

Characterization of the rice blast resistance gene Pik cloned from Kanto51

To study similar, but distinct, plant disease resistance (R) specificities exhibited by allelic genes at the rice blast resistance locus Pik/Pikm, we cloned the Pik gene from rice cultivar Kanto51 and compared its molecular features with those of Pikm and of another Pik gene cloned from cv. Kusabue. Like Pikm, Pik is composed of two adjacent NBS-LRR (nucleotide-binding site, leucine-rich repeat) genes: the first gene, Pik1-KA, and the second gene, Pik2-KA. Pik from Kanto51 and Pik from Kusabue were not identical; although the predicted protein sequences of the second genes were identical, the sequences differed by three amino acids within the NBS domain of the first genes. The Pik proteins from Kanto51 and Kusabue differed from Pikm in eight and seven amino acids, respectively. Most of these substituted amino acids were within the coiled-coil (CC) and NBS domains encoded by the first gene. Of these substitutions, all within the CC domain were conserved between the two Pik proteins, whereas all within the NBS domain differed between them. Comparison of the two Pik proteins and Pikm suggests the importance of the CC domain in determining the resistance specificities of Pik and Pikm. This feature contrasts with that of most allelic or homologous NBS-LRR genes characterized to date, in which the major specificity determinant is believed to lie in the highly diverged LRR domain. In addition, our study revealed high evolutionary flexibility in the genome at the Pik locus, which may be relevant to the generation of new R specificities at this locus.     

Differential requirement of Oryza sativa RAR1 in immune receptor-mediated resistance of rice to Magnaporthe oryzae

The required for Mla12 resistance (RAR1) protein is essential for the plant immune response. In rice, a model monocot species, the function of Oryza sativa RAR1 (OsRAR1) has been little explored. In our current study, we characterized the response of a rice osrar1 T-DNA insertion mutant to infection by Magnaporthe oryzae, the causal agent of rice blast disease. osrar1 mutants displayed reduced resistance compared with wild type rice when inoculated with the normally virulent M. oryzae isolate PO6-6, indicating that OsRAR1 is required for an immune response to this pathogen. We also investigated the function of OsRAR1 in the resistance mechanism mediated by the immune receptor genes Pib and Pi5 that encode nucleotide binding-leucine rich repeat (NB-LRR) proteins. We inoculated progeny from Pib/osrar1 and Pi5/osrar1 heterozygous plants with the avirulent M. oryzae isolates, race 007 and PO6-6, respectively. We found that only Pib-mediated resistance was compromised by the osrar1 mutation and that the introduction of the OsRAR1 cDNA into Pib/osrar1 rescued Pib-mediated resistance. These results indicate that OsRAR1 is required for Pib-mediated resistance but not Pi5-mediated resistance to M. oryzae.     

Direct interaction of resistance gene and avirulence gene products confers rice blast resistance

Rice expressing the Pi-ta gene is resistant to strains of the rice blast fungus, Magnaporthe grisea, expressing AVR-Pita in a gene-for-gene relationship. Pi-ta encodes a putative cytoplasmic receptor with a centrally localized nucleotide-binding site and leucine-rich domain (LRD) at the C-terminus. AVR-Pita is predicted to encode a metalloprotease with an N-terminal secretory signal and pro-protein sequences. AVR-Pita(176) lacks the secretory and pro-protein sequences. We report here that transient expression of AVR-Pita(176) inside plant cells results in a Pi-ta-dependent resistance response. AVR-Pita(176) protein is shown to bind specifically to the LRD of the Pi-ta protein, both in the yeast two-hybrid system and in an in vitro binding assay. Single amino acid substitutions in the Pi-ta LRD or in the AVR-Pita(176) protease motif that result in loss of resistance in the plant also disrupt the physical interaction, both in yeast and in vitro. These data suggest that the AVR-Pita(176) protein binds directly to the Pi-ta LRD region inside the plant cell to initiate a Pi-ta-mediated defense response.     

Molecular diversity in rice blast resistance gene Pi-ta makes it highly effective against dynamic population of Magnaporthe oryzae

Rice blast is one of the important diseases of rice which can be effectively managed by the deployment of resistance genes. Pi-ta is one of the major blast resistant genes effective against pathogen populations in different parts of India. We analysed allelic variants of Pi-ta from 48 rice lines selected after phenotyping of 529 rice landraces across three eco-geographical blast hot spot regions. Besides, Pi-ta orthologue sequences of 220 rice accessions belonging to wild and cultivated species of rice were also included in the study for a better evo–devo perspective of the diversity present in the gene and the selection pressures acting on this locus. We obtained high nucleotide variations (SNPs and insertion–deletions) in the intronic region. We also identified 64 haplotypes based on nucleotide polymorphism in these alleles. Pi-ta orthologues of Indian landraces were scattered in eight major haplotypes indicating its heterogenous nature. We identified a total of 47 different Pi-ta protein variants on the basis of deduced amino acid residues amongst the orthologues. Five unique and novel Pi-ta variants were identified for the first time in rice landraces exhibiting different reaction types against the Magnaporthe oryzae population. A high value of Pi_non/syn was observed only in the leucine-rich domain of the alleles cloned from Indian landraces, indicating strong selective forces acting on this region. The detailed molecular analysis of the Pi-ta orthologues provides insights to a high degree of inter- and intraspecific relationships amongst the Oryza species. We identified rice landraces possessing the effective alleles of this resistance gene which can be used in future blast resistance breeding programmes.