Identification And Characterization Of Rice Blast Resistance Gene Pid4 In Rice Digu

Rice blast caused by the fungal pathogen Magnaporthe oryzae is the most destructive disease of rice,which seriously threatens the rice yield and quality.Digu,a Chinese indica rice variety,confers robust and durable resistance to all of the tested blast isolates.Digu has been long and widely used as an important genetic resource for blast resistance breeding.However,the genetic basis that underlies the durable BSR of Digu is still not fully understood.In this study,we conducted a comparative transcriptomic profiling of Digu and LTH(a susceptible variety)at 5,10,and 20 hours post-inoculation(hpi)of M.oryzae,and coupled with genome-wide sequence analysis.In this way,we isolated a new blast resistance(R)gene,Pid4.The main results are summarized as below:1.Through the comparative transcriptomic analysis,we identified nine Digu-specific expressed NBS-LRR genes from 511 probe sets that correspond to annotated NBS-LRR proteins.By comparing the genomic sequences between Digu and 66 non-durably resistant rice accessions,we identified seven SNPs located in either exons or promoter regions of NBS-LRR genes from 2576 Digu-specific SNPs.Among them,there was one Digu-specific SNP corresponding to the gene,NBS2(LOC_Os06g17950),which was also identified by comparative transcriptomic profiling.2.Genomic sequence and expression pattern analyses revealed that the NBS2 in Digu encodes a complete NBS-LRR protein and is expressed stably,whereas the NBS2 in LTH is a pseudogene and remains undetectable by qRT-PCR analysis.As both of the RILs(recombinant inbred lines)and transgenic lines carrying NBS2-Digu displayed resistance to M.oryzae isolates,we named this NBS-LRR gene Pid4.Blast resistant tests showed that Pid4 confers resistance to both leaf and neck blast diseases,and Pid4 is different from Pi-z,Piz-t,Piz-5,and Pi9 in resistance spectrum.3.The spatio-temporal expression pattern analysis of Pid4 showed that,Pid4 was expressed at all developmental stages and all tissues tested.The strongest expression was detected from leaves and stems at the booting stage,whereas relatively weak expression was detected in roots at the booting stage.In addition,the expression of Pid4 was not obviously responsive to infection by either compatible or incompatible M.oryzae isolate.4.Pid4 contains a 2,148 bp coding region,a 587 bp 5'UTR and a 1,508 bp 3'UTR.The N-terminus of the Pid4 protein contains two potential coiled-coil(CC)domains;the NBS domain contains five typical motifs,viz P-loop,WalkerB,Kinase 3a,GLPL,and RNBS-D;the C-terminus is the LRR region that comprises six irregular LRR repeats.Thus the Pid4 gene encodes a typical CC-NBS-LRR protein.When transiently expressed in rice protoplasts,the PID4-GFP fusion protein was distributed in both nucleus and cytoplasm.5.Phylogenetic tree analysis showed that Pid4 belongs to a same Clade with Pi2,Piz-t,Pigm,Pi50,and Pi9.The pairwise amino acid sequence alignment suggested that Pi2,Piz-t,Pigm,Pi50,and Pi9 shares high sequence identity(>95%)to each other,whereas Pid4 shares low sequence identity(~52%)to each of them.Pid4 is located in a gene cluster containing 14 NBS-LRR genes and 3 transposons in Digu.In this cluster,except for Pid4,the other 13 NBS-LRR genes show a one-to-one correspondence(Identity > 99%)with the 13 NBS-LRR genes in Pigm cluster.In the Pigm(or Pi2,Pi9)cluster,the gene that shares highest sequence similarity to Pid4 is Pigm-R12(or Pi2-R12,Pi9-R12),rather than the reported R genes in those clusters.These results suggest that Pid4 is a novel R gene identified in this cluster.6.The sequence diversity analysis of the Pid4 alleles showed that,the NBS domain of the alleles in different varieties is conserved,whereas the sequence polymorphism is mainly concentrated in promoter region and LRR domain.As Nipponbare,TP309,and LTH are three susceptible varieties,we did a sequence alignment of pid4-NIP,pid4-LTH,and pid4-TP309.The result showed that,all the three alleles contain a 1-bp deletion(+1294,A)that leads to a premature termination and consequent a truncated polypeptide,suggesting the most likely reason for their loss of function.In addition,the expression level of Pid4 alleles in different varieties was not directly related to their resistance level.7.Further molecular analysis indicated that 10% of 210 rice varieties genotyped by the Pid4 marker may contain the Pid4 locus.According to the classification of the tested varieties,the Pid4-containing varieties were mainly distributed in Africa and belonged to the tropical japonica,whereas the selection degree of Pid4 was limited in Asian and indica varieties.These results can provide reference for the application of Pid4 in rice blast resistant breeding.Here,we demonstrate the effectiveness of integrating transcriptome and genome-wide sequence based strategy in quickly isolating plant R genes.This strategy can also be used to identify genes with polymorphisms in regulatory regions or splice sites that often contribute to phenotypic diversity in plant resistance.The identification and characterization of Pid4 may lead to a better understanding of the resistance mechanism of Digu and facilitate the application of R genes in resistant breeding.