The Blast Resistance Gene Pi54of Cloned from Oryza officinalis Interacts with Avr-Pi54 through Its Novel Non-LRR Domains

The dominant rice blast resistance gene Pi54 cloned by map-based cloning approach from indica rice cultivar Tetep confers broad spectrum resistance to Magnaporthe oryzae. In this investigation, an orthologue of Pi54 designated as Pi54of was cloned from Oryza officinalis conferring high degree of resistance to M. oryzae and is functionally validated. We have also characterized the Pi54of protein and demonstrate its interaction with AVR-Pi54 protein. The Pi54of encoded ∼43 kDa small and unique cytoplasmic LRR family of disease resistance protein having unique Zinc finger domain overlapped with the leucine rich repeat regions. Pi54of showed Magnaporthe-induced expression. The phylogenetic and western blot analysis confirmed orthologous nature of Pi54 and Pi54of genes, whereas the identity of protein was confirmed through MALDI-TOF analysis. The in silico analysis showed that Pi54of is structurally more stable than other cloned Pi54 proteins. The molecular docking revealed that Pi54of protein interacts with AVR-Pi54 through novel non-LRR domains such as STI1 and RhoGEF. The STI1 and GEF domains which interact with AVR-Pi54 are also components of rice defensome complex. The Pi54of protein showed differential domain specificity while interacting with the AVR protein. Functional complementation revealed that Pi54of transferred in two rice lines belonging to indica and japonica background imparts enhanced resistance against three highly virulent strains of M. oryzae. In this study, for the first time, we demonstrated that a rice blast resistance gene Pi54of cloned from wild species of rice provides high degree of resistance to M. oryzae and might display different molecular mechanism involved in AVRPi54-Pi54of interaction.     

Molecular Mapping of Rice Blast Resistance Gene Pi-k h in the Rice Variety Tetep

We have used rice line Tetep as a resistant donor with the aim of mapping a durable blast resistance gene Pi-k ^h using RAPD and AFLP techniques in conjunction with bulk segregant analysis. An F₂ mapping population consisting of 205 plants was generated by crossing Tetep with HP2216, a highly susceptible cultivar. Inoculation with specific isolate (PLP-1) of Magnaporthe grisea at seeding stage showed that the Pi-k ^h gene inherited as a single dominant gene in F2 population. RAPD analysis was performed with 240 primers to detect polymorphism between resistant and susceptible parents. Of these, 48 primers produced polymorphic banding pattern between resistant and susceptible parents. Bulk segregant analysis was performed with 48 primers of which 5 showed polymorphism between resistant and susceptible bulks. A 700 bp DNA band was obtained in resistant F2 plants with primer 5-129 indicating its linkage to the resistance gene. Out of 64 AFLP primer combinations used for polymorphism survey between HP 2216 and Tetep, 11 AFLP primer combinations were able to distinguish the resistant and susceptible bulks. An AFLP band of 75 bp obtained with primer combination, E-TAlM-CTC co-segregated with the resistance gene. The RAPD marker 5-129₇₀₀ and AFLP₇₅ were placed on the linkage map at a distance of 2.1 eM and 15.1 eM flanking to Pi-k ^hgene, respectively. The RAPD band closely linked to Pi-k ^h gene was sequenced and used for the development of CAPs markers which also co-segregated with resistant phenotype in the mapping population. On sequence analysis and homology search of RAPD fragment with whole rice genome sequence database and the information available on physical, genetic and sequence maps of rice, the co-segregating CAPs marker was placed at long arm of rice chromosome 11. CAPs marker developed in this study showed polymorphism in different rice cultivars grown in North-Western Himalayan region and is being used for the pyramiding of Pi-k ^h gene along with other blast resistance genes using marker-assisted selection.     

High-resolution mapping, cloning and molecular characterization of the Pi-k h gene of rice, which confers resistance to Magnaporthe grisea

In order to understand the molecular mechanisms involved in the gene-for-gene type of pathogen resistance, high-resolution genetic and physical mapping of resistance loci is required to facilitate map-based cloning of resistance genes. Here, we report the molecular mapping and cloning of a dominant gene (Pi-k ^h ) present in the rice line Tetep, which is associated with resistance to rice blast disease caused by Magnaporthe grisea. This gene is effective against M. grisea populations prevalent in the Northwestern Himalayan region of India. Using 178 sequence tagged microsatellite, sequence-tagged site, expressed sequence tag and simple sequence repeat (SSR) markers to genotype a population of 208 F₂ individuals, we mapped the Pi-k ^h gene between two SSR markers (TRS26 and TRS33) which are 0.7 and 0.5 cM away, respectively, and can be used in marker-assisted-selection for blast-resistant rice cultivars. We used the markers to identify the homologous region in the genomic sequence of Oryza sativa cv. Nipponbare, and a physical map consisting of two overlapping bacterial artificial chromosome and P1 artificial chromosome clones was assembled, spanning a region of 143,537 bp on the long arm of chromosome 11. Using bioinformatic analyses, we then identified a candidate blast-resistance gene in the region, and cloned the homologous sequence from Tetep. The putative Pi-k ^h gene cloned from Tetep is 1.5 kbp long with a single ORF, and belongs to the nucleotide binding site-leucine rich repeat class of disease resistance genes. Structural and expression analysis of the Pi-k ^h gene revealed that its expression is pathogen inducible.     

Broad-spectrum Blast Resistance Gene Pi-k h Cloned from Rice Line Tetep Designated as Pi54

Blast disease caused by Magnaporthe oryzae is one of the important biotic stresses of rice. So far more than 85 blast resistance genes have been identified of these more than 14 have already been cloned. A broad spectrum rice blast resistance gene Pi-k ^h was cloned from the rice line Tetep. The gene was named Pi-k ^h based on the earlier reports on its genetic analysis in various rice lines. However, with the advances in molecular genetics and genomics of rice, the Pik locus has now been mapped more precisely. Since there are two reports on the mapping of Pi-k ^h gene from different rice lines, there is some confusion in the naming of this gene. In this report the name of Pi-k ^h gene cloned from the rice line Tetep has been designated as per the standard guidelines of Committee on Gene Symbolization, Nomenclature and Linkage (CGSNL) and its physical location on rice chromosome 11, which is ～2.5 Mbp away from the Pik locus mapped recently. Hence Pi-k ^h gene cloned from Tetep is now designated as Pi54.     

Gene interactions and genetics of blast resistance and yield attributes in rice (Oryza sativa L.)

Blast disease caused by the pathogen Pyricularia oryzae is a serious threat to rice production. Six generations viz., P₁, P₂, F₁, F₂, B₁ and B₂ of a cross between blast susceptible high-yielding rice cultivar ADT 43 and resistant near isogenic line (NIL) CT13432-3R, carrying four blast resistance genes Pi1, Pi2, Pi33 and Pi54 in combination were used to study the nature and magnitude of gene action for disease resistance and yield attributes. The epistatic interaction model was found adequate to explain the gene action in most of the traits. The interaction was complementary for number of productive tillers, economic yield, lesion number, infected leaf area and potential disease incidence but duplicate epistasis was observed for the remaining traits. Among the genotypes tested under epiphytotic conditions, gene pyramided lines were highly resistant to blast compared to individuals with single genes indicating that the nonallelic genes have a complementary effect when present together. The information on genetics of various contributing traits of resistance will further aid plant breeders in choosing appropriate breeding strategy for blast resistance and yield enhancement in rice.     

Improvement of seedling and panicle blast resistance in <Emphasis Type="Italic">Xian</Emphasis> rice varieties following <Emphasis Type="Italic">Pish</Emphasis> introgression

Rice blast is a serious disease caused by the filamentous ascomycetous fungus Magnaporthe oryzae. Incorporating disease resistance genes in rice varieties and characterizing the distribution of M. oryzae isolates form the foundation for enhancing rice blast resistance. In this study, the blast resistance gene Pish was observed to be differentially distributed in the genomes of rice sub-species. Specifically, Pish was present in 80.5% of Geng varieties, but in only 2.3% of Xian varieties. Moreover, Pish conferred resistance against only 23.5% of the M. oryzae isolates from the Geng-planting regions, but against up to 63.2% of the isolates from the Xian-planting regions. Thus, Pish may be an elite resistance gene for improving rice blast resistance in Xian varieties. Therefore, near-isogenic lines (NILs) with Pish and the polygene pyramid lines (PPLs) PPL^Pish/Pi1, PPL^Pish/Pi54, and PPL^Pish/Pi33 in the Xian background Yangdao 6 were generated using a molecular marker-assisted selection method. The results suggested that (1) Pish significantly improved rice blast resistance in Xian varieties, which exhibited considerably improved seedling and panicle blast resistance after Pish was introduced; (2) PPLs with Pish were more effective than the NILs with Pish regarding seedling and panicle blast resistance; (3) the PPL seedling and panicle blast resistance was improved by the complementary and overlapping effects of different resistance genes; and (4) the stability of NIL and PPL resistance varied under different environmental conditions, with only PPL^Pish/Pi54 exhibiting highly stable resistance in three natural disease nurseries (Jianyang, Jinggangshan, and Huangshan). This study provides new blast resistance germplasm resources and describes a novel molecular strategy for enhancing rice blast resistance.     

Detection of novel blast resistance genes, Pi58(t) and Pi59(t), in a Myanmar rice landrace based on a standard differential system

The use of broad-spectrum R genes is an effective way to achieve durable resistance against rice blast (Magnaporthe oryzae Couch, anamorph: Pyricularia oryzae Cavara) in rice (Oryza sativa L.). We previously surveyed the diversity of blast resistance in 948 rice varieties and found a Myanmar rice landrace, Haoru (International Rice Research Institute genebank acc. no. IRGC33090), with broad-spectrum resistance against the standard differential blast isolates. Here, we examined the genetic basis of Haoru’s broad-spectrum resistance by using the standard blast differential system consisting of the standard isolates and differential varieties. For genetic analysis, we used a BC1F1 population and BC1F2 lines derived from crosses of Haoru with a susceptible variety, US-2. Co-segregation analysis of the reaction pattern in the BC1F1 population against the 20 standard isolates suggested that Haoru harbors three R genes. By using bulk-segregant and linkage analysis, we mapped two of the three R genes on chromosomes 12 and 6, and designated them as Pi58(t) and Pi59(t), respectively. Pi58(t) and Pi59(t) were differentiated from other reported R genes using the standard differential system. The estimated resistance spectrum of Pi58(t) corresponded with that of Haoru, suggesting that Pi58(t) is primarily responsible for Haoru’s broad-spectrum resistance. In addition, Pi59(t) and the third gene were also proven to be new and useful genetic resources for studying and improving blast resistance in rice.     

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.     

Sequence variation at the rice blast resistance gene Pi-km locus: Implications for the development of allele specific markers

The recently cloned blast resistance (R) gene Pi-km protects rice crops against specific races of the fungal pathogen Magnaporthe oryzae in a gene-for-gene manner. The use of blast R genes remains the most cost-effective method for an integrated disease management strategy. To facilitate rice breeding we developed a Pi-km specific DNA marker. For this purpose, we initially explored the existing sequence diversity for alleles of the two genes responsible for the Pi-km specificity. The analysis of 15 rice cultivars revealed that the majority of nucleotide polymorphisms were associated with the Pi-km1 gene. Interestingly, the correspondent amino acid variation was localized within the predicted coiled-coil domain of the putative Pi-km1 protein. In contrast, the sequence of Pi-km2 alleles was highly conserved even within distantly related cultivars. Furthermore, disease reactions of the selected cultivars to five M. oryzae isolates, as well as their determined Pi-km1 allele, showed a good correlation with the known Pi-k genes (-k/-kh/-km/-ks/-kp) historically reported for these cultivars. Based on these findings, specific primer sets have been designed to discriminate among the various Pi-km alleles. The new markers should simplify the introgression of the valuable blast resistance associated with the complex Pi-k locus into rice cultivars.     

Development and validation of functional marker targeting an InDel in the major rice blast disease resistance gene Pi54 (Pik h )

Rice blast is one of the most devastating diseases affecting the rice crop throughout the world. In molecular breeding for host plant resistance, functional markers are very useful for enhancing the precision and accuracy in marker-assisted selection (MAS) of target gene(s) with minimum effort, time and cost. Pi54 (which was earlier known as Pik ^h ) is one of the major blast resistance genes and has been observed to show resistance against many isolates of the blast pathogen in India. The gene has been cloned through map-based strategy and encodes a nucleotide-binding site?Cleucine-rich repeat (NBS?CLRR) domain-containing protein. In the present study, we carried out allele mining for this gene and identified a 144-bp insertion/deletion (InDel) polymorphism in the exonic region of the gene. A PCR-based co-dominant molecular marker targeting this InDel, named Pi54 MAS, was developed. Pi54 MAS was observed to perfectly co-segregate with blast resistance in a mapping population with no recombinants. Validation of this marker in 105 genotypes which are either susceptible or resistant to rice blast disease showed that the marker is polymorphic in most of the resistant?Csusceptible genotype combinations and is more accurate than the earlier reported markers for Pi54. Hence this functional, co-dominant marker is suggested for routine deployment in MAS of Pi54 in breeding programs.     

Expression of a plant defensin in rice confers resistance to fungal phytopathogens

Transgenic rice (Oryza sativa L. cv. Pusa basmati 1), overexpressing the Rs-AFP2 defensin gene from the Raphanus sativus was generated by Agrobacterium tumefaciens-mediated transformation. Expression levels of Rs-AFP2 ranged from 0.45 to 0.53% of total soluble protein in transgenic plants. It was observed that constitutive expression of Rs-AFP2 suppresses the growth of Magnaporthe oryzae and Rhizoctonia solani by 77 and 45%, respectively. No effect on plant morphology was observed in the Rs-AFP2 expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of Rs-AFP2 plants on the in vitro growth of M. oryzae indicated that the Rs-AFP2 protein produced by transgenic rice plants was biologically active. Transgene expression of Rs-AFP2 was not accompanied by an induction of pathogenesis-related (PR) gene expression, suggesting that the expression of Rs-AFP2 directly inhibits the pathogens. Here, we demonstrate that transgenic rice plants expressing the Rs-AFP2 gene show enhanced resistance to M. oryzae and R. solani, two of the most important pathogens of rice.     

Identification of SSR markers for a broad-spectrum blast resistance gene Pi20(t) for marker-assisted breeding

The Pi20(t) gene was determined to confer a broad-spectrum resistance against diverse blast pathotypes (races) in China based on inoculation experiments utilizing 160 Chinese Magnaporthe oryzae (formerly Magnaporthe grisea) isolates, among which isolate 98095 can specifically differentiate the Pi20(t) gene present in cv. IR24. Two flanking and three co-segregating simple sequence repeat (SSR) markers for Pi20(t), located near the centromere region of chromosome 12, were identified using 526 extremely susceptible F₂ plants derived from a cross of Asominori, an extremely susceptible cultivar, with resistant cultivar IR24. The SSR OSR32 was mapped at a distance of 0.2 cM from Pi20(t), and the SSR RM28050 was mapped to the other side of Pi20(t) at a distance of 0.4 cM. The other three SSR markers, RM1337, RM5364 and RM7102, co-segregated with Pi20(t). RM1337 and RM5364 were found to be reliable markers of resistance conditioned by Pi20(t) in a wide range of elite rice germplasm in China. As such, they are useful tags in marker-assisted rice breeding programs aimed at incorporating Pi20(t) into advanced rice breeding lines and, ultimately, at obtaining a durable and broad spectrum of resistance to M. oryaze. Wei Li and Cailin Lei contributed equally to this work.     

Intercellular Production of Tamavidin 1, a Biotin-Binding Protein from Tamogitake Mushroom,Confers Resistance to the Blast Fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in Transgenic Rice

The blast fungus Magnaporthe oryzae, one of the most devastating rice pathogens in the world, shows biotin-dependent growth. We have developed a strategy for creating disease resistance to M. oryzae whereby intercellular production of tamavidin 1, a biotin-binding protein from Pleurotus cornucopiae occurs in transgenic rice plants. The gene that encodes tamavidin 1, fused to the sequence for a secretion signal peptide derived from rice chitinase gene, was connected to the Cauliflower mosaic virus 35S promoter, and the resultant construct was introduced into rice. The tamavidin 1 was accumulated at levels of 0.1–0.2% of total soluble leaf proteins in the transgenic rice and it was localized in the intercellular space of rice leaves. The tamavidin 1 purified from the transgenic rice was active, it bound to biotin and inhibited in vitro growth of M. oryzae by causing biotin deficiency. The transgenic rice plants showed a significant resistance to M. oryzae. This study shows the possibility of a new strategy to engineer disease resistance in higher plants by taking advantage of a pathogen’s auxotrophy.     

Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.)

A gene derived from grapevine (Vitis vinifera) coding for stilbene synthase has been transferred into protoplasts of the commercially important japonica rice cultivar Nipponbare using PEG-mediated direct gene transfer. Transgenic plants were regenerated from calli selected on kanamycin. Southern blot analysis of genomic DNA isolated from regenerants and progeny plants demonstrated that the stilbene synthase gene is stably integrated in the genome of transgenic rice plants and inherited in the offspring. The transient formation of stilbene-synthase-specific mRNA shortly after inoculation with the fungus of the rice blast Pyricularia oryzae has demonstrated that the grapevine stilbene synthase promoter is also active in monocotyledonous plants. Preliminary results indicate an enhanced resistance of transgenic rice to P. oryzae. Received: 1 July 1996 / Revision received: 5 November 1996 / Accepted: 30 November 1996     

Marker-aided selection and validation of various $${ }$$ gene combinations for rice blast resistance in elite rice variety ADT 43

Rice blast caused by fungal pathogen Pyricularia oryzae has a major impact on reducing yield potential of rice. In this study, homozygous plants were selected using microsatellite markers from the \(\hbox {BC}_{3}\hbox {F}_{2}\) population pyramided with four major genes in elite rice variety ADT 43. Background and selected lines with various blast resistance gene combinations were screened under natural conditions to study the effects of various gene combinations. Upon inspection of lines with different gene combinations, the three-gene pyramided line Pi54+Pi33+Pi1 was found to be highly resistant with the score of 3.3 followed by other three-gene pyramided lines Pi54+Pi2+Pi1 and Pi33+Pi2+Pi1, with the scores of 3.9 and 3.8, respectively. Two-gene pyramided lines Pi54+Pi1, Pi33+Pi1 and Pi2+Pi1 were found to be moderately resistant with a mean score of 4.0 each. In the case of monogenic lines, positive plants for Pi54 performed almost equal to three-gene pyramided lines with a mean score of 3.6. Lines with Pi2 and Pi1 were found to be moderately resistant and moderately susceptible with the mean scores of 4.1 and 4.5, respectively.     

Rice blast resistance gene Pikahei-1(t), a member of a resistance gene cluster on chromosome 4, encodes a nucleotide-binding site and leucine-rich repeat protein

Pikahei-1(t) is the strongest quantitative trait locus (QTL) for blast resistance in upland rice cv. Kahei, which has strong field resistance to the rice blast disease. A high-quality bacterial artificial chromosome library was used to fine-map Pikahei-1(t) within ~300 kb on the 31-Mb region on rice chromosome 4. Of the 42 predicted open reading frames, seven resistance gene analogs (RGAs) with the nucleotide-binding site and leucine-rich repeat (NBS-LRR) domain were identified. Among these, RGA1, 2, 3, 5, and 7, but not RGA4 and 6, were found to be expressed in Kahei and monogenic lines containing Pikahei-1(t). Blast inoculation of transgenic rice lines carrying the genomic fragment of each RGA revealed that only RGA3 was associated with blast resistance. On the basis of these results, we concluded that RGA3 is the Pikahei-1(t) and named it Pi63. Pi63 encoded a typical coiled-coil-NBS-LRR protein and showed isolate-specificity. These results suggest that Pi63 behaves like a typical Resistance (R) gene, and the strong and broad-spectrum resistance of Kahei is dependent on natural pyramiding of multiple QTLs. The blast resistance levels of Pi63 were closely correlated with its gene expression levels, indicating a dose-dependent response of Pi63 function in rice resistance. Pi63 is the first cloned R gene in the R gene cluster on rice chromosome 4, and its cloning might facilitate genomic dissection of this cluster region.