Heterogeneous evolutionary rates of Pi2/9 homologs in rice

ABSTRACT: BACKGROUND: The Pi2/9 locus contains multiple nucleotide binding site--leucine-rich repeat (NBS-LRR) genes in the rice genome. Although three functional R-genes have been cloned from this locus, little is known about the origin and evolutionary history of these genes. Herein, an extensive genome-wide survey of Pi2/9 homologs in rice, sorghum, Brachypodium and Arabidopsis, was conducted to explore this theme. RESULTS: In our study, 1, 1, 5 and 156 Pi2/9 homologs were detected in Arabidopsis, Brachypodium, sorghum and rice genomes, respectively. Two distinct evolutionary patterns of Pi2/9 homologs, Type I and Type II, were observed in rice lines. Type I Pi2/9 homologs showed evidence of rapid gene diversification, including substantial copy number variations, obscured orthologous relationships, high levels of nucleotide diversity or/and divergence, frequent sequence exchanges and strong positive selection, whereas Type II Pi2/9 homologs exhibited a fairly slow evolutionary rate. Interestingly, the three cloned R-genes from the Pi2/9 locus all belonged to the Type I genes. CONCLUSIONS: Our data show that the Pi2/9 locus had an ancient origin predating the common ancestor of gramineous species. The existence of two types of Pi2/9 homologs suggest that diversifying evolution should be an important strategy of rice to cope with different types of pathogens. The relationship of cloned Pi2/9 genes and Type I genes also suggests that rapid gene diversification might facilitate rice to adapt quickly to the changing spectrum of the fungal pathogen M. grisea. Based on these criteria, other potential candidate genes that might confer novel resistance specificities to rice blast could be predicted.     

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.     

Genomic structure and evolution of the Pi2/9 locus in wild rice species

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is a devastating disease of rice worldwide. Among the 85 mapped resistance (R) genes against blast, 13 have been cloned and characterized. However, how these genes originated and how they evolved in the Oryza genus remains unclear. We previously cloned the rice blast R-genes Pi2, Pi9, and Piz-t, and analyzed their genomic structure and evolution in cultivated rice. In this study, we determined the genomic sequences of the Pi2/9 locus in four wild Oryza species representing three genomes (AA, BB and CC). The number of Pi2/9 family members in the four wild species ranges from two copies to 12 copies. Although these genes are conserved in structure and categorized into the same subfamily, sequence duplications and subsequent inversions or uneven crossing overs were observed, suggesting that the locus in different wild species has undergone dynamic changes. Positive selection was found in the leucine-rich repeat region of most members, especially in the largest clade where Pi9 is included. We also provide evidence that the Pi9 gene is more related to its homologues in the recurrent line and other rice cultivars than to those in its alleged donor species O. minuta, indicating a possible origin of the Pi9 gene from O. sativa. Comparative sequence analysis between the four wild Oryza species and the previously established reference sequences in cultivated rice species at the Pi2/9 locus has provided extensive and unique information on the genomic structure and evolution of a complex R-gene cluster in the Oryza genus.     

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.     

水稻广谱抗稻瘟病基因研究进展

稻瘟病是水稻生产中的最严重病害之一,由于稻瘟菌小种的高度变异性,垂直抗性基因难以持续控制稻瘟病的危害,因此,克隆和利用广谱持久抗瘟基因被认为是解决稻瘟病问题最经济有效的策略。本文从广谱抗源的筛选与利用,广谱抗瘟基因的定位、克隆与应用等方面对水稻广谱抗稻瘟病基因研究取得的进展进行了概述,并介绍了广谱抗性分子机理的最新研究进展。基于国内外稻瘟病抗性基因研究的现状及趋势,以及我国丰富的抗瘟水稻种质资源,克隆越来越多的广谱抗瘟基因具有重要的理论与应用价值。     

Genetic characterization and fine mapping of the blast resistance locus Pigm(t) tightly linked to Pi2 and Pi9 in a broad-spectrum resistant Chinese variety

The identification and utilization of broad-spectrum resistance genes have been proven the most effective and economical approach to control rice blast disease. To understand the molecular mechanism of broad-spectrum resistance to rice blast, we conducted genetic and fine mapping analysis of the blast resistance gene in a Chinese rice variety: Gumei 4 (GM4) identified with broad-spectrum resistance and used in rice breeding for blast resistance for more than 20 years. Genetic and mapping analysis indicated that blast resistance to nine isolates of different Chinese races in GM4 was controlled by the same dominant locus designated as Pigm(t) that was finely mapped to an approximately 70-kb interval between markers C5483 and C0428 on chromosome 6, which contains five candidate NBS--LRR disease resistance genes. The allelism test showed that Pigm(t) was either tightly linked or allelic to Pi2 and Pi9, two known blast resistance genes. Mapping information also indicated that another blast resistance gene Pi26(t) might also be located at the same region. Candidate genes were identified by sequence analysis of the Nipponbare and Pi9 locus and the corresponding region in GM4. Sequence divergence of candidate genes was observed between GM4 and model varieties Nipponbare and 9311, and Pi9. Our current study provides essential information and new genetic resource for the cloning of functional resistance gene(s) and for marker-assisted selection in rice breeding for broad-spectrum blast resistance.Yiwen Deng and Xudong Zhu contributed equally to this work.     

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.     

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.     

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.     

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.     

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.     

Identification and fine mapping of <Emphasis Type="Italic">Pi33</Emphasis>, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene <Emphasis Type="Italic">ACE1</Emphasis>

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) × Azucena (susceptible) and Azucena × Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F₂ populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.Communicated by D.J. Mackill     

SSR Markers Closely Linked to the Pi-z Locus are Useful for Selection of Blast Resistance in a Broad Array of Rice Germplasm

Pi-z is a disease resistance gene that has been effectively used to combat a broad-spectrum of races of the rice blast fungus Magnaporthe grisea. Although DNA markers have been reported for selection of the Pi2(t) and Pi-z resistance genes at the Pi-z locus, markers that are more tightly linked to the Pi-z locus would benefit rapid and effective cultivar development. Analysis of the publicly available genome sequence of Nipponbare near the Pi-z locus revealed numerous SSRs that could be converted into markers. Three SSRs on rice PAC AP005659 were found to be very tightly linked to the Pi-z locus, with one marker, AP5659-3, co-segregating with the Pi-z resistance reaction. The Pi-z factor conferring resistance to two races of blast was mapped to a 57 kb region on the physical map of Nipponbare in a location where the Pi2(t) gene was physically mapped. Two SSR marker haplotypes were unique for cultivars carrying the Pi-z gene, which indicates these markers are useful for selection of resistance genes at the Pi-z locus in rice germplasm.     

Identification and fine mapping of a major R gene to Magnaporthe oryzae in a broad-spectrum resistant germplasm in rice

Identification of R genes and development of associated molecular markers will facilitate their application in the development of crop cultivars resistant to disease. We evaluated the resistance of a resistant germplasm ??D69??, 10 monogenic lines, and model cultivar ??Nipponbare?? to 56 M. oryzae isolates of blast disease in rice. The results demonstrated that only D69 exhibited full-spectrum resistance among the 12 investigated materials. Resistance inheritance in D69 was analyzed using a stable isolate GD08T13 with strong pathogenicity, collected from diseased panicles. A single dominant R gene was revealed and designated as Pi51(t). Through linkage analysis and the development of new markers, Pi51(t) was subsequently delimited to an interval of ~100.8?kb flanked by markers Ind306 and RM19818, where Pi2, Pi9, Piz, Piz-t, Pigm(t), and Pi40(t) reside. Different genotypes identified by linked markers pB8, Pi9-2, zt56591, and T845, and different pathotypes to the same set of isolates, distinguished Pi51(t) from Pi2, Pi9, Piz, and Piz-t. The origin of Pi40(t) in wild rice suggests that Pi51(t) and Pi40(t) are different. Comparison of resistance spectra suggests multiple R genes in D69, making its resistance durable and valuable in breeding programs. The results of this work will facilitate future studies on cloning and functional analysis of blast resistance genes for rice improvement.     

Development of near-isogenic lines with different alleles of Piz locus and analysis of their breeding effect under Yangdao 6 background

Rice blast is one of the most destructive diseases of rice. The most effective way of managing this disease is to develop resistant cultivars by introducing resistance genes into elite rice recipients. In this study, the near-isogenic lines (NILs) of six resistance alleles of the Piz locus (Pizt, Pi2, Pigm, Pi40, Pi9 and Piz) were constructed with Yangdao 6 as genetic background. Seedling inoculation tests showed that most of the NILs, namely NIL-Pi2, NIL-Pigm, NIL-Pi9, NIL-Pizt and NIL-Pi40, exhibited good resistance to blast with resistance frequencies (RFs) of over 82.50 %, execpt NIL-Piz which showed lower resistance with a RF of only 36.13 %. Furthermore, the improved-resistance NILs exhibited high similarity of their resistance spectra, with overlapping degrees of resistance spectrum (OD) of more than 75.83 %. However, the RF of panicle blast for all NILs decreased significantly compared with seedling blast in an artificial inoculation test. Although NIL-Pigm showed a higher panicle blast RF of 80 %, other NILs with outstanding performance in seedling blast resistance, namely NIL-Pizt, NIL-Pi2, NIL-Pi9 and NIL-Pi40, exhibited middle or low RFs of panicle blast with values from 56.67 to 33.30 %. Natural induction in a disease nursery showed a consistent trend in artificial inoculation results at seedling and heading stages. While NIL-Pigm was found to exhibit good resistance to leaf blast and panicle blast, NIL-Pi9 and NIL-Pizt were further demonstrated to show excellent resistance in Suichuan, Jiangxi province and Enshi, Hubei province, respectively, because of the race–region specificity. Agronomic traits of NILs were also investigated in order to evaluate the linkage drag effect of different alleles of the Piz locus. The resistance effects of the different alleles of the Piz locus under identical genetic background against seedling blast and panicle blast was first reported in this study, and the above results are expected to provide a theoretical support for the rational utilization of broad-spectrum resistance genes in breeding practice.     

Marker-assisted breeding of Chinese elite rice cultivar 9311 for disease resistance to rice blast and bacterial blight and tolerance to submergence

Rice (Oryza sativa L.) is the staple food crop for more than half of the world’s population. The development of hybrid rice is a practical approach to increase rice production. However, rice production was frequently affected by biotic and abiotic stresses. Rice blast and bacterial blight are two major diseases in rice growing regions. Rice plantation is also frequently affected by short-term submergence or seasonal floods in wet seasons and drought in dry seasons. The utilization of natural disease resistance (R) genes and stress tolerance genes in rice breeding is the most economic and efficient way to combat or adapt to these biotic and abiotic stresses. Rice cultivar 9311 is widely planted rice variety, either as inbred rice or the paternal line of two-line hybrid rice. Here, we report the pyramiding of rice blast R gene Pi9, bacterial blight R genes Xa21 and Xa27, and submergence tolerance gene Sub1A in 9311 genetic background through backcrossing and marker-assisted selection. The improved rice line, designated as 49311, theoretically possesses 99.2% genetic background of 9311. 49311 and its hybrid rice, GZ63S/49311, conferred disease resistance to rice blast and bacterial blight and showed tolerance to submergence for over 18 days without significant loss of viability. 49311 and its hybrids had similar agronomic traits and grain quality to 9311 and the control hybrid rice, respectively. The development of 49311 provides an improved paternal line for two-line hybrid rice production with disease resistance to rice blast and bacterial blight and tolerance to submergence.