Allele Mining for Blast Resistance in Rice (PDF)

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Plant Stress 12 (2024) 100465 Contents lists available at ScienceDirect Plant Stress journal homepage: www.sciencedirect.com/journal/plant-stress Allele mining for blast-resistance gene at Pi5 locus in rice Ying Zhou b, 1, Yi Xu b, 1, Xue Wang a, Shuyan Kou c, Ping Huang c, Wenxiu Qiu b, d, Liu Duan a, *, Li Liu a, * a State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, School of Life Sciences, Hubei University, Wuhan 430062, Hubei, China b College of Life Science and Health, Wuhan University of Science and Technology, Wuhan 430065, Hubei, China, c Institute of Food Crops, Yunnan Academy of Agricultural Sciences, Kunming, Yunnan 650205, China d Wuhan Towin Biotechnology Co., Ltd., Wuhan 430073, China A R T I C L E I N F O A B S T R A C T Keywords: Rice plays a crucial role in global food security, serving as a staple for more than half of the world’s population. Pi5 Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most devastating fungal diseases, resulting in Nucleotide diversity substantial economic losses in terms of rice yield and grain quality. The resistance provided by individual blast Rice blast resistance genes tends to become ineffective within a few years of extensive agricultural use due to the rapid Resistance gene alleles Crop breeding evolution of the fungus. Therefore, continuous efforts in resistance breeding in rice are required to enrich the pool of new resistance genes and alleles. A large-scale screening of new blast resistance alleles was conducted across 2000 rice accessions from major rice-producing areas in China. Field trials were employed in two rice uniform blast nurseries located in Enshi and Yichang. Approximately 153 rice accessions showed at least moderate resistance to the rice blast. Sequence-based allele mining was used to identify the allelic variants of major rice blast resistance genes at the Pi5 locus of chromosome 9. Six novel alleles were identified from 64 accessions. Field tests at five locations, including Enshi, Yichang, Lvtian, Taojiang, and Yaan, and greenhouse inoculation using 33 blast strains from Hubei, Hunan, Guangzhou, and Sichuan, were employed to verify the resistance of these six alleles. Three near-isogenic lines (types 2, 4, and 6) were constructed and inoculated with 31 blast isolates from Heilongjiang to evaluate their blast resistance. Furthermore, bulk segregant analysis was used to confirm that Pi5-type2 and Pi5-type6 are dominant alleles in the analyzed populations. These novel alleles expand the allelic series, enriching the genetic resources for rice blast resistance breeding programs, especially in the north of China. Furthermore, they provide valuable insights into the molecular interactions between rice and rice blast. 1. Introduction 2015; Zhu et al., 2016; Wang et al., 2017; Sahu et al., 2022a). The monoculture of crop varieties over large areas and the rapid evolution of Rice blast, caused by the fungus Magnaporthe oryzae (M. oryzae) predominant pathogenic races pose significant challenges to rice pro­ (Synonym: Pyricularia oryzae), is one of the most devastating diseases duction. M. oryzae, which is known for its genetic instability and path­ prevalent in rice fields, leading to substantial yield losses in epidemi­ ogenic variability, often leads to the breakdown of resistance within a cally favorable areas and seasons. The disease, including leaf and panicle few years (Jiang et al., 2012). Plants have evolved diverse mechanisms blast, can result in yield reductions ranging from 10 % to 30 % in to protect themselves against pathogen invasion and colonization in severely affected crops (Xie et al., 2019). Consequently, harnessing host response to pathogen threats. Previous studies have revealed that most resistance has been considered the most effective and economic resistance genes encode receptor proteins with nucleotide-binding sites approach for disease control (Yadav et al., 2019). After years of relent­ and leucine-rich repeats (NBS-LRR) (Wang et al., 2018), organized in less efforts by scientists, more than 500 loci and 146 R genes have been tight complex clusters with multiple copies. Extensive genetic studies of identified, with 37 of them molecularly characterized (Ashkani et al., the rice blast resistance genes revealed that nine of the thirteen major * Corresponding authors. E-mail addresses: [email protected] (L. Duan), [email protected] (L. Liu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.stress.2024.100465 Received 5 January 2024; Received in revised form 9 April 2024; Accepted 21 April 2024 Available online 22 April 2024 2667-064X/© 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). Y. Zhou et al. Plant Stress 12 (2024) 100465 genes were clustered (Wu et al., 2012), and most of the genes are MR-HR against field mix-inoculum were selected for molecular broad-spectrum resistance genes with viable resistance. A lot of resis­ screening. Lijiangxin Tuan Heigu (LTH), which is highly susceptible to tance genes were mapped at the same location, close to the centromere rice blast, was used as a control for disease evaluation. The leading of chromosome 9, and four R-genes from this locus (Pii, Pi3, Pi5, and japonica variety in Northeast China, Kongyu 131 (KY131), was used as a Pi15) have been cloned (Dai et al., 2010). Genomic analysis of this locus cross parent for RICE6K-based genotyping and bulk segregant analysis in rice cultivars and wild rice lines has shown that the copy number and and also as the recurrent parent to construct near-isogenic lines at the SNP genotyping were variable in Pii homologs, indicating a complex Pi5 locus for rice blast resistance genotyping analysis. evolutionary history for this R-gene locus that involved processes (Wu et al., 2012). The clustered arrangement has been implicated in the 2.2. Pathogen collection, inoculation, and disease evaluation evolution of novel resistance specificities through processes such as gene conversion, recombination, or unequal crossing over (Dai et al., 2010). For resistance spectrum analysis, 64 blast isolates of different races Some NBS-LRR gene homologs located at the same locus exhibited a were used in this study: 33 from the southern rice region (Hubei, Hunan, different evolutionary pattern. However, little is known about the Sichuan, and Guangdong) and 31 from the northeastern rice region in evolutionary patterns within the Pi5 locus. In order to gain a further Heilongjiang. All these isolates were collected from major rice-growing understanding of the origin and evolutionary history of this locus, provinces in China, have genetic differentiation, and belong to different genomic sequences of Pi5 homologs from 153 rice blast-resistance va­ blast lineages. The 64 Magnaporthe oryzae isolates, which are highly rieties, which were collected from more than 2000 varieties, were virulent on most of the rice lines, were used to analyze the phenotypes of selected for evolutionary analyses. Our findings suggest the involvement parents with disease resistance genes and for phenotypic analysis of the of this locus in adaptation. It is important to recognize that the diverse Pi5 genotype. Two week-old seedlings were spray-inoculated with spore evolutionary patterns observed among Pi5 homologs reflect a complex suspensions (1 × 105 spores/ml) in a dew growth chamber for 24 h in evolutionary history. darkness at 26◦ C and were subsequently kept at 12/12 h (day/night), Resistance genes (Pii, Pi3, Pi5, and Pi15) from different rice cultivars 26◦ C, and 90 % humidity. After 7 days post-inoculation, the disease have been mapped to the long arm of chromosome 9 (Takagi et al., reaction of each rice line was recorded using a 0–5 disease rating scale. 2013). It has been shown that the four R genes, Pii, Pi3, Pi5, and Pi15 (Lin et al., 2007), confer broad-spectrum resistance (Yi et al., 2004; Lin et al., 2.3. PCR for allele mining and blast resistance genes 2007; Tian et al., 2022). Pii was shown to confer resistance against 10 isolates collected from China, Thailand, and Japan. In a test involving 10 After field rice blast resistance identification was carried out on over isolates, lines containing Pii exhibited resistance to 5 isolates (Qi et al., 2000 rice accessions in Enshi and Yichang, the leaves of resistant ma­ 2020). Pi15 is located in the same region as a cluster of multiple terials above the moderately resistant (MR) level were taken for DNA NBS-LRR genes, NBS-Pi15, a solo member of the Pii gene cluster, confers extraction. For designing PCR primers, the rice genomic sequence of the broad-spectrum resistance to rice blast and exhibited resistance to eight Oryza sativa (O. sativa) cv. Nipponbare (www.ncbi.nlm.nih.gov), isolates (Tian et al., 2022). While all these genes bestow broad-spectrum O. sativa cv. Fujisaka5 (MH490982.1 and MH490981.1), O. sativa cv. resistance against rice blast, their resistance specificities have been F036 (OL689233.1 and OL689234.1), O. sativa cv. BJ (OL689231.1 and observed to differ from one another. The molecular basis of this locus in OL689232.1), and O. sativa cv. Hitomebore (AB820896.1 and conferring broad-spectrum blast resistance, however, remains to be LC190730.1), corresponding to the location of the Pi5 locus genes, were elucidated. Despite being mapped at the same location, these 3 genes used. Two sets of primers flanking the full-length gene were designed exhibit different tandem repeats, possibly due to their distinct evolu­ using the Primer 3 software (http://primer3.ut.ee/). The internal tionary origins. To delve into the molecular intricacies of this locus, we overlapping oligos were designed for sequencing using the primer conducted sequencing on various resistance materials. The results walking technique. PCR amplification of alleles was done using genomic revealed six novel alleles in this region, each demonstrating notable DNA extracted from rice leaves using the modified CTAB method differences in sequences. Through the analysis of flanking sequences and (Murray and Thompson, 1980). The PCR reaction of 50 μl was setup resistance identification in these materials, we observed significant with 50 ng of template DNA, 50 ng both forward and reverse primers, 5 variations in the resistance cluster spectrum. Employing Bulk Segregant μL 10 × LA Taq Buffer II, 8 μL dNTP Mixture (2.5 mM each), and 2.5 U Analysis (BSA) with allele donor materials and susceptible controls, we TaKaRa LA Taq (RR02MQ, TAKARA). The details of the various primers identified three regions associated with resistance localized at the Pi5 used in the present study are given in Table S1. locus. This implies that different Pi5 alleles may manifest different blast Amplified PCR products were purified and sequenced using Sanger’s resistance spectra. According to these results, the new resistance alleles dideoxy method on an automated capillary-based DNA Analyzer might offer prospects for their utilization in breeding programs aimed at Sequencer, ABI 3730XL (ABI, Applied Biosystems, Amersham, USA). enhancing rice blast resistance. Each allele was sequenced twice using all the sequencing primers. The sequence read length was around 800 nucleotides, so primer walking 2. Materials and methods was performed to fill the gap in order to obtain full-length gene sequences. 2.1. Plant materials 2.4. DNA sequence analysis Over 2000 rice accessions were assembled, encompassing 1214 core germplasm resources primarily in landrace form from Huazhong Agri­ All the sequence reads generated for each allele by the forward, cultural University, 514 varieties from Heilongjiang Institute of Agri­ reverse, and internal primers were assembled separately for each allele cultural Sciences, and over 200 varieties sourced from institutions by using Sequencing Analysis Software Version 5.1 (Applied Bio­ including the United States Department of Agriculture and China Na­ systems). The high-quality sequence was assembled, and the consensus tional Rice Research Institute. Rice blast-resistant varieties were sequence was used in the analysis. The assembled DNA sequence of each measured through natural inducement in five rice uniform blast nurs­ allele was used to do Blast2Sequences analysis against the Pi5 genes to eries, which are located at Enshi and Yichang in Hubei province, Lvtian check its purity. in Guangdong province, Yaan in Sichuan province, and Taojiang in The Pi5 genes from the rice blast-resistant lines were used as a Hunan province. Based on the standard scoring system for leaf blast reference for comparison studies. Structural analyses of Pi5 alleles were (scale HS-HR, HS: Highly Susceptible; HR: Highly Resistant) (Zhou et al., performed to predict gene structure and ORF region using FGENESH 2018), the varieties that were resistant with a phenotypic score of software trained for monocot (www.softberry.com). The data were 2 Y. Zhou et al. Plant Stress 12 (2024) 100465 recorded as the number of exons and their positions, as well as the Province, China. presence of polyA tails and SNPs within the ORF regions. For Single Following the International Rice Research Institute (IRRI) classifi­ Nucleotide Polymorphism (SNP) search within the ORFs, multiple cation criteria for rice blast, disease resistance is categorized into six sequence alignment was done for all the alleles along with Pii and Pi5 classes: HR (high resistance), R (resistance), MR (medium resistance), alleles as references by using the Software of Sequencer. InDels and SNPs MS (medium sensitivity), S (sensitivity), and HS (high sensitivity). were identified in the form of transitions and transversions. The protein Through our evaluation, we identified 153 accessions exhibiting resis­ sequences of predicted ORFs were utilized for motif scanning using the tance phenotypes (HR, R or MR) in Enshi or Yichang. A PCR-based motif scan tool. For motif scan PROSITE patterns, PROSITE, HAMAP screening was employed to detect the presence of Pii and Pi5, and we profile, Pfam, and HMMS (local and global models) were used as pa­ identified 64 accessions as candidates for allele mining. rameters. Multiple sequence alignments of DNA sequences from ampli­ fied gene fragments and protein sequences from the ORF region of all the 3.2. Isolation of Pi5 alleles alleles along with that of Pi5 and Pii of control rice lines, were done by using CLUSTALW (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and PCR amplification was conducted on the 153 rice accessions exhib­ MEGA11.0 (http://www.megasoftware.net) software. The parameters iting the rice blast resistance phenotype. Out of these, sequences employed in MEGA included 1000 replications for bootstrap analysis encompassing the entire coding region of the Pi5 locus, from the start and the neighbour-joining method with the p - distance model. A codon to the stop codon, were successfully amplified in 64 samples. phylogenetic tree was constructed to study the evolution of the Pi5 Through sequence analysis, six novel Pi5 alleles, which named Pi5- allele. Additionally, the software developed by our group was employed Type1 to Pi5-Type6, were identified within these 64 varieties. A com­ to display the differences between the DNA sequences of all alleles and parison was conducted between the obtained sequences and the re­ the reference sequences. ported Pi5 gene sequence, with a specific focus on the Pi5–1 and Pi5–2 loci. These loci represent the two most crucial tandem repeat regions 2.5. RICE6K-based genotyping and bulk segregant analysis (BSA) associated with rice blast resistance. Notably, these six new alleles are unique due to the presence of unique SNPs/InDels within their se­ For the obtained materials containing the novel Pi5 alleles, the quences. Fig. 1 illustrates a schematic diagram depicting the alignment resistance to rice blast was identified by natural onset in the field in five of the identified Pi5 allele sequences. units and inoculated in the greenhouse with 33 blast strains. Accessions exhibiting a resistance rate exceeding 80 % were selected and subjected 3.3. Sequence analysis of Pi5 alleles to hybridization with KY131, resulting in the generation of F2 progeny. For each population, approximately 200 individual rice F2 plants were Six distinct alleles were identified, each exhibiting variations from inoculated and identified for rice blast, with one strain collected from the reference Pi5 gene in terms of nucleotide polymorphisms, insertions, Heilongjiang. The traditional BSA strategy was conducted to prepare and deletions, either uniquely or shared among the different alleles. At genomic DNA pools for analysis. DNA samples from 20 plants displaying the Pi5–1 locus, two alleles (Pi5-Type4 and Pi5-Type5) contain complete the highest resistance were selected for DNA extraction to create the R- ORF structures, while at the Pi5–2 locus, three alleles (Pi5-Type1, Pi5- pool. Additionally, DNA from 20 plants exhibiting the highest suscep­ Type4, and Pi5-Type5) contain complete ORF structures. Pi5-Type3, the tibility was extracted to form the S-pool. one with the lowest homology, lacks a complete ORF structure at both RICE6K, a whole-genome single nucleotide polymorphism (SNP) the Pi5–1 and Pi5–2 loci. Pi5-Type4 and Pi5-Type5, housing complete array (Yu et al., 2014), was used for genetic background profiling of the ORFs at the Pi5–1 locus, share identical sequences with Pi5–1, still donor Pi5 lines. DNA amplification, fragmentation, chip hybridization, display many Snps/InDels at the Pi5–2 site. In contrast to the conser­ single base extension, staining, and scanning were conducted by the Life vative nature of the Pi5–1 locus, higher variability among different al­ Science and Technology Center of China National Seed Group Co., Ltd. leles is observed at the Pi5–2 locus (Table 1). (Wuhan, China), according to the Infinium HD Assay Ultra Protocol (http://www.illumina.com/). 3.4. Analysis of predicted Pi5 proteins Both R-pool and S-pool, together with their parent DNA, underwent RICE6K-based genotyping and bulk segregant analysis to determine the Protein-level analysis of Pi5 proteins reveals that Pi5 alleles consist of resistance locus. About 6′000 known SNP markers from RICE6K three conserved domains, including CC, NBS, and LRR. Two of these distributing on the 12 chromosomes of rice were screened in the parents alleles, Pi5-Type4 and Pi5-Type5, possess complete open reading frames and the two bulks. The likely polymorphic markers associated with the (ORF) similar to that of Pi5. Conversely, the remaining four alleles (Pi5- trait were used to genotype the F2 population to confirm the possible Type1, Pi5-Type2, Pi5-Type3, and Pi5-Type6) exhibit shorter ORFs due resistance locus. SSR primers were then screened using the parents and to sequence variations leading to premature termination codons. Among the two bulk DNA samples, from which some primer combinations the four alleles, the sequence of Pi5-Type1 maintains complete ORFs of revealed bands that were polymorphic not only among parental geno­ Pi5–1, but with a deletion in Pi5–1. On the other hand, the sequences of types but also between the bulk DNA pair. Based on the evaluation of the Pi5-Type2, Pi5-Type3, and Pi5-Type6 have some deletions in both Pi5–1 DNA bulks, individual F2 plants were analyzed with cosegregating and Pi5–2, resulting in translated proteins shorter than that of Pii (Fig. 1, primers to confirm SNP marker linkage to resistance. Table 1). 3. Results 3.5. Phylogeny and distribution of new Pi5 allele in rice subspecies 3.1. Assessment of disease phenotypes in over 2000 rice accessions with As four known broad-spectrum resistance genes, Pii, Pi3, Pi5, and M. oryzae Pi15, were all located in the same region, about 9.67 Mb on the long arm of chromosome 9, six alleles of Pi5 were sequenced using the sequencing A comprehensive collection of more than 2000 rice accessions were primers (Table S1). collected from the primary rice-growing provinces of China. These To understand the genetic relatedness among the Pi5 alleles, a collection encompasses two major subspecies: indica (approximately phylogenetic analysis of the six alleles, the cloned Pi5 gene, Pii, Pi2, 75 %) and japonica (approximately 25 %). To evaluate the blast Pigm, Piz-t, Pi9, Piks, Pikp, and Pikm was conducted. Nucleotide se­ phenotype of these accessions, rice plants were cultivated in regions quences, including the complete ORFs, were utilized to construct the with high incidences of rice blast, namely Enshi and Yichang in Hubei phylogenetic tree (Fig. 2). Three major clusters were observed. Cluster I 3 Y. Zhou et al. Plant Stress 12 (2024) 100465 Fig. 1. Schematic maps for sequence alignment of novel Pi5 alleles, Pi5-1 (A) and Pi5-2 (B) genes. Schematic diagram of gene structure was shown above, black boxes indicate exons and white boxes indicate introns. The start codon and the stop codon are labeled with ATG and TGA, respectively. Six Pi5 alleles isolated from the studied rice materials, along with three published Pii alleles, were compared with the Pi5-1 (A) and Pi5-2 (B) genes, the unit scale indicates the location of nu­ cleotides. The black line on the bar indicates the amino acid polymorphism compared with the reference sequence. The gap between allele strips indicates deletion, and the size of gap indicates the length of deletion sequence. Table 1 Single nucleotide polymorphisms (SNPs) and different alleles of Pi5 gene in various rice cultivars. Pi5 allele Accessiona Identity to Identity to Sequence complete or complete Number of Snps/ Number of Number of Pi5–1 Pi5–2 not InDels SNPs InDels Pi5–1 Pi5–2 Pi5–1 Pi5–2 Pi5–1 Pi5–2 Pi5 XS09 – – Yes Yes Yes – – – – – Pii Hitomebore 100 % 96 % Yes Yes Yes 25 0 22 0 3 Pii- Fujisaka 99 % 42 % Yes Yes Yes 52 22 19 6 5 Fujisaka Pii-F036 F036 99 % 52 % Yes Yes Yes 53 26 15 7 5 Pii-BJ BJ 99 % 52 % Yes Yes Yes 20 5 9 1 5 Pi5-Type1 KAUKKYI 25 % 46 % No No Yes 33 5 21 1 6 ANI Pi5-Type2 ZH233 24 % 28 % No No No 59 35 18 3 3 Pi5-Type3 YGSM 24 % 21 % No No No 97 46 42 5 4 Pi5-Type4 R03138 100 % 46 % Yes Yes Yes 27 0 20 0 7 Pi5-Type5 BG90–2 100 % 46 % Yes Yes Yes 18 0 12 0 6 Pi5-Type6 YD4038 24 % 42 % No No No 84 46 32 4 2 a Representative donor accession containing the allele. SNP, Single nucleotide polymorphism; InDel, Insertion and deletion. comprised Pi5–1 alleles, whereas cluster II encompassed Pi5–2 alleles. susceptible control (CK1), were measured through natural inducement Notably, the tandem repeat region at the Pi9 locus formed cluster II- in five rice uniform blast nurseries, which are located at Enshi and subgroup I, including Pi2, Pi9, Pigm, and Piz-t. Furthermore, alleles Yichang in Hubei province, Taojiang in Hunan province, Yaan in from the Pik locus were distributed across subgroups II and III within Sichuan province, and Lvtian in Guangdong province (Table 2). The cluster II (Fig. 2). donor varieties with the Pi5-Type1, Pi5-Type2, Pi5-Type4, and Pi5- Type6 alleles showed better resistance. Meanwhile, the leaf blast resis­ tance of these rice accessions was assessed in greenhouse using 33 iso­ 3.6. Evaluation of blast resistance against M. oryzae lates of M. oryzae from Hubei, Jiangxi, Hunan, Fujian, and Guangdong provinces in the south of China (Table 3). XS09, which harbors the Pi5 To evaluate the blast resistance of these six novel Pi5 alleles against donor, exhibited broad-spectrum resistance to rice blast, with a resis­ M. oryzae, accessions with novel Pi5 alleles: KAUKKYI ANI (Pi5-Type1), tance frequency of 90.63 % against M. oryzae isolated from the south of ZH233(Pi5-Type2), YGSM (Pi5-Type3), R03138 (Pi5-Type4), BG90-2 China. The alleles of Pi5 donors showed a resistance frequency ranging (Pi5-Type5), YD4038 (Pi5-Type6), along with XS09 (Pi5) and LTH as 4 Y. Zhou et al. Plant Stress 12 (2024) 100465 Fig. 2. Phylogenetic relationship among gDNA of NBS genes at Pi5 locus. Six novel Pi5 allele sequences found in our research materials and all NBS disease resistance genes at the tandem repeat region of the Pik, Pi2/9 and Pi5 locus were used for analysis. Phylogenetic tree of the genomic sequences for 6 novel Pi5 alleles based on our sequencing results and NBS genes at Pik, Pi2/9 and Pi5 locus. Bootstrap values (1000 replications) are mentioned at the branch nodes. from 53.13 % to 96.88 %. Accessions ZH233, R03138, and YD4038, from Heilongjiang province in the north of China (Table 4). The results containing the Pi5-Type2, Pi5-Type4, and Pi5-Type6 alleles, were showed that donors harboring Pi1, Pi2, Pi9, Pigm, and Pi5 genes resistant against more than 29 out of 33 blast isolates, with a resistance exhibited resistance frequencies ranging from 3.23 % to 96.77 % against frequency of over 87.50 % (Table 3). M. oryzae from Heilongjiang. The alleles of Pi5 donors exhibited resis­ Therefore, ZH233 (Pi5-Type2), R03138 (Pi5-Type4), and YD4038 tance frequencies ranging from 67.74 % to 96.77 %. Furthermore, The (Pi5-Type6) were selected for crosses with KY131 (as the recurrent NILs containing Pi5-Type2, Pi5-Type4, and Pi5-Type6 haplotypes parent) to generate near-isogenic lines at the Pi5 locus. The leaf blast demonstrated resistance against more than 19 out of 31 blast isolates, resistance of the three BC2F2 NILs, six alleles of Pi5 donors (Pi5-Type1 to with a resistance frequency exceeding 61.29 % (Table 4). Pi5-Type6), Pi1, Pi2, Pi9, Pigm, and Pi5, along with LTH as the suscep­ tible control, was assessed in a greenhouse using 31 isolates of M. oryzae 5 Y. Zhou et al. Plant Stress 12 (2024) 100465 Table 2 result in distinct prevailing strains across different regions (Tolstrup Disease responses of cloned genes and Pi5 allele honor plants in field text. et al., 1997). At the same time, due to the robust and rapid variability of Gene Accessiona Enshi Yichang Lvtian Yaan Taojiang physiological race of rice blast pathogens, a single resistance gene can be easily overcome by the physiological races of M. oryzae (Lee et al., 2009; CK1 LTH HS HS HS HS HS Pi5 XS09 HR HR R HR MS Li et al., 2017). Therefore, identifying and cloning novel genes that Pi5-Type1 KAUKKYI ANI HR R R HR MS confer broad-spectrum and highly resistance to rice blast becomes Pi5-Type2 ZH233 HR HR MR HR R paramount under such variable conditions (Mi et al., 2018; Zhou et al., Pi5-Type3 YGSM S S MR S HS 2018). In this study, a large-scale screening across 2000 rice accessions Pi5-Type4 R03138 HR HR MR HR MS Pi5-Type5 BG90-2 R HS MR S MS identified 153 rice accessions with moderate to high blast resistance, Pi5-Type6 YD4038 HR HR MR HR MS and six novel alleles at the Pi5 locus were identified and validated a through field trials and greenhouse inoculation. The exploration of al­ Representative donor accessions containing the alleles, which were used as leles at key loci of important disease resistance genes using a large and parents. S, Sensitivity; MS, Medium sensitivity; HS, High sensitivity; R, Resis­ tance; MR, Medium resistance; HR, High resistance. diverse set of rice germplasm resources, particularly involving indica and japonica, facilitates the identification of novel alleles contributing to broad-spectrum resistance and enables the development of rice va­ 3.7. Blast resistance mediated by the Pi5 allele locus rieties with enhanced disease resistance, ultimately contributing to global food security. To elucidate the genetic relationship between the Pi5 allele genes With the development of molecular marker technology, the con­ and rice blast resistance, F2 populations were generated through crosses struction of high-density genetic linkage maps, and the improvement of between three donor rice varieties harboring Pi5 alleles (Pi5-Type2, Pi5- related molecular techniques, more than 500 loci and 146 R genes have Type4, and Pi5-Type6, respectively) and the susceptible japonica vari­ been identified, with 37 of them molecularly characterized (Ashkani ety, KY131. Approximately 200 individuals from each F2 population et al., 2015; Zhu et al., 2016; Wang et al., 2017; Sahu et al., 2022b). Pi5 were inoculated with a M. oryzae strain isolated from Heilongjiang was discovered through genetic mapping and positional cloning strate­ (M15Ga006). For each population, DNA samples from 20 plants dis­ gies, and is a broad-spectrum resistance gene that confers resistance to playing the highest resistance were selected for DNA extraction to create the rice blast pathogen, M. oryzae (Xiao et al., 2020). With the exception the R-pool, and DNA from 20 plants exhibiting the highest susceptibility of a limited number of rice blast resistance genes, the predominant was extracted and mixed to form the S-pool. The RICE6K whole-genome structure among resistance genes is the NBS-LRR configuration, which single nucleotide polymorphism (SNP) array was used to analyze the two parental lines, R-pool, and S-pool (Fig. 3). With around 60′000 SNP markers in the RICE6K array, the mean value of Q30 reached 91.92 %, Table 4 ensuring high-quality data and the accuracy of the bulk segregant Disease responses of cloned genes and Pi5 allele honor plants to 31 Magnaporthe analysis. Pi5-type2, Pi5-type4, and Pi5-type6 are localized within the oryzae isolates from Heilongjiang Province of China. 9.67 Mb region on the long arm of chromosome 9, proximal to the Gene Generation Accession Resistance ratio Resistance tandem repeat region. CK1 Parent LTH 0 8.90±0.30 CK2 Parent KY131 3.23 % 7.45±1.83 4. Discussion Pi5 Parent XS09 96.77 % 0.81±1.65 Pi5-Type1 Parent KAUKKYI ANI 67.74 % 2.84±2.48 Rice blast, caused by the fungus M. oryzae, is one of the most sig­ Pi5-Type2 Parent ZH233 77.42 % 3.03±2.57 nificant diseases affecting rice globally (Ashkani et al., 2015; Kong et al., Pi5-Type3 Parent YGSM 87.10 % 1.90±1.84 2023). The disease has been found in nearly all rice-producing regions Pi5-Type4 Parent R03138 96.77 % 0.97±1.13 Pi5-Type5 Parent BG90–2 83.87 % 2.71±2.19 worldwide (Zhu et al., 2000; Miah et al., 2013). In China, the annual Pi5-Type6 Parent YD4038 77.42 % 2.65±2.86 disease area exceeds 3.8 million hectares, with a loss of 1 billion kilo­ Pi1 BC3F2 CT9506 3.23 % 7.26±1.75 grams of rice yield (Zeng et al., 2009). Although chemical control can Pi2 BC3F2 C101A51 64.52 % 3.45±2.33 reduce diseases and yield losses to some extent, it has greatly increased Pi9 BC3F2 75–1–127 96.77 % 1.87±1.17 Pigm BC3F2 C1185 90.32 % 1.84±1.58 the burden on farmers and caused great environmental pollution. Pi5 BC2F2 XS09 80.65 % 2.58±2.04 Therefore, the optimal approach to preventing rice blast involves the Pi5-Type2 BC2F2 ZH233 64.52 % 3.32±2.24 development of resistant varieties through breeding efforts. The utili­ Pi5-Type4 BC2F2 R03138 74.19 % 2.58±1.90 zation of molecular marker-assisted selection for the incorporation of Pi5-Type6 BC2F2 YD4038 61.29 % 3.65±2.42 rice blast resistance genes into susceptible materials represents a sig­ a Representative donor accession containing the allele. Inoculated with 31 nificant advancement in breeding, and this approach has been widely M. oryzae isolates from Heilongjiang Province. The average disease score inoc­ used in breeding. However, variations in locations and environments ulated with 31 M. oryzae isolates. Table 3 Disease responses of cloned genes and Pi5 allele honor plants to 33 Magnaporthe oryzae isolates from south of China. Gene Accessiona Hubei Hunan Sichuan Guangdong Total Resistance Resistance ratio CK1 LTH 8.38±1.12 8.50±1.12 9.00±0.00 9.00±0.00 8.61±0.92 0.00 % Pi5 XS09 1.50±0.18 1.50±0.35 1.50±0.41 4.00±1.91 2.05±1.36 90.63 % Pi5-Type1 KAUKKYI ANI 4.47±2.94 3.20±2.39 5.00±1.73 2.64±1.07 3.94±2.48 71.88 % Pi5-Type2 ZH233 1.72±0.88 1.50±0.00 3.88±3.54 1.50±0.29 1.91±1.48 96.88 % Pi5-Type3 YGSM 3.50±2.00 5.30±1.64 3.38±1.44 7.57±1.34 4.66±2.39 53.13 % Pi5-Type4 R03138 2.44±1.44 2.70±1.10 2.00±1.00 3.71±2.31 2.70±1.62 87.50 % Pi5-Type5 BG90–2 2.53±1.55 2.20±1.57 2.00±1.00 4.00±1.91 2.73±1.67 78.13 % Pi5-Type6 YD4038 1.50±0.37 1.90±0.89 1.50±0.41 3.79±1.87 2.06±1.32 96.88 % a Representative donor accession containing the allele. Inoculated with 33 M. oryzae isolates from south of China. The average disease score inoculated with 33 M. oryzae isolates. 6 Y. Zhou et al. Plant Stress 12 (2024) 100465 Fig. 3. Haplotype maps of genetic background profiling of using RICE6K array. F2 population resulting from the cross of Pi5-Type2, 4, and 6 A with KY131 were used for the analysis. The red box indicates the area that produces resistance on chromosome 9. R: the extreme resistance pool containing DNA samples from 20 plants displaying the highest resistance. S: extreme susceptible pool, DNA from 20 plants exhibiting the highest susceptibility was extracted and mixed. Each short line at the chromosomes indicates the position of a single nucleotide polymorphism (SNP) showing the differences between R (or S) and R&S in common. AA, female parental homozygous genotype; BB, male parental homozygous genotype; and AB, heterozygous genotype. includes Pi5. Studying the allelic variations of disease resistance genes et al., 2011). In our study, we investigated the polymorphism of the Pi5 provides crucial insights into the generation and specificity of novel allele in 153 rice accessions, including Pi5–1 and Pi5–2. Our results resistance genes. Previous reports showed that Pii is an allele of Pi5 by support their conclusion, showing higher variability among different DNA sequence characterization and complementation analysis, and both alleles at the Pi5–2 locus compared to Pi5–1. The presence of SNPs and of them require the presence of two nucleotide-binding leucine-rich InDels between them suggests lower selection pressure in these regions, repeat genes arranged in series for rice blast resistance (Vo et al., 2019a). indicating that Pi5 experiences more selection pressure at the Pi5–1 Studying the inherent diversity within R genes will enable the detection locus. As previous results showed that Pi5–1 transcripts accumulate after of essential amino acid residues responsible for recognition specificity in pathogen challenge, whereas the Pi5–2 gene is constitutively expressed R proteins and the unearthing of new R alleles. In a previous study, (Lee et al., 2009), it suggests that they play different roles in the defense nucleotide variation in nine R genes was examined across 13 indica and response of rice against rice blast disease. Pi5-Type4 and Pi5-Type5 are 13 japonica rice cultivars, as well as 12 accessions of wild rice (Liu et al., completely identical to Pi5 at the Pi5–1 site, and there are complete gene 2011). The findings revealed a mean nucleotide diversity of 3.2 %, with sequences at the Pi5–2 site, both of which have 46 % identity to Pi5–2. Pi5–1 being categorized as relatively conserved among rice R genes (Liu However, their resistance spectrum to different rice blast strains behaves 7 Y. Zhou et al. Plant Stress 12 (2024) 100465 distinctly. Therefore, we conclude that while Pi5–1 remains conserved, across different rice-growing regions revealed greater strain diversity in the occurrence of mutations in Pi5–2 alone can lead to changes in southern areas (Zhang et al., 2017). This diversity is representative of resistance spectrum. Evidence from sites such as Yaan shows that adaptations to the ecological environment of different regions, including Pi5-Type4 and Pi5 exhibit HR-resistant phenotypes, while Pi5-Type5 is climate, moisture, and soil conditions, determines the varied rice pro­ susceptible. Similarly, in Yichang, Pi5-Type4 and Pi5 display HR resis­ duction patterns of China’s rice production ecological zones (Zhang tance phenotypes, while Pi5-Type5 shows susceptibility to HS. Similar to et al., 2017). Guangdong Province, characterized by a temperate climate Pi5, other rice blast R genes such as Pike and Pikm comprise two adjacent and ample rainfall, predominantly cultivates indica rice (Hang et al., CC–NBS-LRR genes and are required for resistance (Chen et al., 2015). 2023). Hunan and Hubei provinces have higher temperatures in summer However, unlike Pi5, the first genes (e.g., Pike-1 for Pike and Pikm-1 for and lower temperatures in autumn, and both indica and japonica rice Pikm) are rich in DNA sequence variation, while the second genes (e.g., varieties are used; Sichuan, with its humid climate, primarily grows Pike-2 for Pike and Pikm-2 for Pikm) are highly conserved (Chen et al., indica rice, while in the cool and dry northeastern province of Hei­ 2015; Vo et al., 2019a). It is assumed that the resistance specificity of longjiang, japonica rice varieties are mainly grown (Zhou and Turvey, these genes may be determined by their first genes rather than their 2014). Notably, Pi1, Pik, Pik-m, and Piz offer significant protection second genes. For example, although two adjacent CC–NBS-LRR genes against blast disease in southern China (Zhang et al., 2017; Zhou et al., (Pike-1 and Pike-2) residing at the Pik locus are required for Pike-­ 2022). However, earlier in northern China, Pi1 was recommended for mediated resistance (Chen et al., 2015), the resistance specificity local breeding programs, Pib and Pik exhibited lower resistance levels mediated by Pike is determined by Pike-1 but not Pike-2, supporting this (Zhang et al., 2017). In our study, however, the inoculation results of Pi1 hypothesis (Meng et al., 2021). However, we cannot rule out the with 31 strains from Heilongjiang revealed unsatisfactory performance contribution of Pi5-1 to disease resistance specificity since we did not in terms of both resistance frequency and level (Zhou et al., 2022). In obtain materials with only mutated Pi5-1 for resistance spectrum testing contrast, we observed that Pi5 and the six newly discovered Pi5 alleles in our study. More evidence, such as transient co-expression in tobacco exhibited relatively high resistance against 33 southern strains and 31 leaves or yeast two-hybrid tests, is needed to elucidate the interaction northern strains. At the same time, the NIL analysis of Pi5-Type2, between Pi5-1/Pi5-2 and its AVR effector. Pi5-Type4, and Pi5-Type6 on KY131, a commonly cultivated rice vari­ Research has revealed that Pii(t), Pi3(t), Pi5(t), and Pi15(t) are all ety in northern China, demonstrated that the newly discovered pi5 al­ located within a common locus on rice chromosome 9 (Sahu et al., leles can enhance the resistance of KY131 against prevalent blast strains 2022a). The resistance spectrum and the genetic linkage between Pi5, in northern China. In addition, the results of RICE6K in this study sug­ Pi3, and Pii genes demonstrate a striking proximity (Jeon et al., 2003; Yi gest that Pi5-type2 and Pi5-type6 are dominant alleles in the analyzed et al., 2004; Selisana et al., 2017). Additionally, the genomic regions in populations. Therefore, our study discovered 6 new Pi5 alleles and lines containing Pi3(t) and Pi5(t) are identical (Jeon et al., 2003). elucidated their potential for rice blast breeding enhancement, which Although the Pii and Pi5 can functionally substitute for each other in will greatly increase rice breading in China especially in the north. initiating effector-triggered immunity (ETI), DNA sequence analysis has uncovered significant differences in the final intron between Pii-2 and 5. Conclusion Pi5-2, resulting in Pii-2 containing an extended C-terminus of 20 amino acids compared to Pi5-2 (Vo et al., 2019b). Moreover, Exo70F3 is not In this study, we investigated more than 2000 rice germplasm re­ required for Pi5-mediated resistance, whereas it is necessary for Pii-­ sources in China, identifying 153 wide-spectrum rice varieties resistant mediated resistance (Vo et al., 2019b). As a result, the hypothesis con­ to rice blast. Subsequent sequencing of the Pi5 allele in these varieties cerning the allelism among the Pi5, Pii, and Pi3 continues to be a matter led to the discovery of six novel alleles of the Pi5 gene, distinct from the of active discussion. The sequence variations observed in NB-LRR genes known Pii, Pi3, Pi5, and Pi15 genes. Three of them showed good resis­ are ascribed to the extensive co-evolution between rice and the rice blast tance to both southern and northern strains. Functional validation, pathogen. Previous studies isolated AVR-Pii through an association ge­ including gene silencing and complementarity experiments, would be netics approach among 23 M. oryzae isolates (Yoshida et al., 2009). helpful in verifying their functionality. These newly identified resistant Correlation between the presence of AVR-Pii and the activation of alleles would be utilized alongside previously cloned blast resistance Pi5-mediated resistance has been found in previous study, however, the genes in field breeding through molecular marker-assisted approaches identification of a few isolates that are avirulent on Pi5 lines despite the to enhance blast resistance durability. absence of AVR-Pii suggests the presence of an additional, yet-to-be-identified AVR-Pi5 (Selisana et al., 2017). The multiallelic CRediT authorship contribution statement resistance loci in our study have provided information on the DNA se­ quences within Pi5 responsible for their distinct resistance specificities, Ying Zhou: Writing – review & editing, Writing – original draft, deepened our understanding of pi5, and laid the groundwork for future Investigation, Data curation. Yi Xu: Writing – review & editing, Writing investigations in monitoring avirulence gene variations, and offered the – original draft, Investigation, Data curation. Xue Wang: Writing – re­ possibility of using them in breeding. view & editing, Visualization. Shuyan Kou: Writing – review & editing. Pyramiding R genes offers opportunities to enhance disease resis­ Ping Huang: Writing – review & editing, Funding acquisition. Wenxiu tance breeding, achieving long-lasting resistance against rice blast. Qiu: Writing – review & editing. Liu Duan: Writing – review & editing, Previous research pointed out that 45 % of the main rice blast resistance Visualization, Supervision, Project administration, Funding acquisition. genes discovered are derived from japonica rice, 51 % are derived from Li Liu: Supervision, Project administration, Funding acquisition. indica rice, and the remaining 4 % are derived from wild rice (Ashkani et al., 2016). Currently, several Pi genes have been utilized to combine two or more R genes, offering superior phenotypic benefits and stability Declaration of competing interest compared to single genes. Examples include Pi1, Pi2, Pi33, Pita, Piz-5, Pi9, Pizt, Pi54, Pid1, and Pib (Samman et al., 1998; Srivastava et al., The authors declare that they have no known competing financial 2017; Chen et al., 2021). Since Pi5 possesses broad-spectrum resistance, interests or personal relationships that could have appeared to influence incorporating it into gene pyramiding would facilitate the breeding of the work reported in this paper. materials with durable resistance to rice blast in China. In our study, more than 2000 rice germplasms were screened and assessed, including Data availability two major subspecies (indica and japonica) from various rice-growing regions. Analysis of the race structure of the Rice Blast Pathogen No data was used for the research described in the article. 8 Y. Zhou et al. Plant Stress 12 (2024) 100465 Acknowlegements Sahu, P.K., Sao, R., Choudhary, D.K., Thada, A., Kumar, V., Mondal, S., Das, B.K., Jankuloski, L., Sharma, D., 2022a. Advancement in the breeding, biotechnological and genomic tools towards development of durable genetic resistance against the This study was suported by the Natural Science Foundation of Hubei rice blast disease. Plants. (Basel) 11. Province (2023AFA016), Key Laboratory of Integrated Management of Sahu, P.K., Sao, R., Choudhary, D.K., Thada, A., Kumar, V., Mondal, S., Das, B.K., Crops of Central China and Hubei Key Laboratory of Crop Disease, Insect Jankuloski, L., Sharma, D., 2022b. Advancement in the breeding, biotechnological and genomic tools towards development of durable genetic resistance against the Pests and Weeds Control (Grant No. 2023ZTSJJ4), National Natural rice blast disease. Plants Basel 11. Science Foundation of China (32300244), and Outstanding Young Samman, I., Wohrle, J.C., Hennerici, M., 1998. Rational therapy of Guillain-Barre Scholar Program of Yunnan Province. syndrome. Lancet 351, 753–754. Selisana, S.M., Yanoria, M.J., Quime, B., Chaipanya, C., Lu, G., Opulencia, R., Wang, G. L., Mitchell, T., Correll, J., Talbot, N.J., Leung, H., Zhou, B., 2017. Avirulence (AVR) Supplementary materials gene-based diagnosis complements existing pathogen surveillance tools for effective deployment of resistance (r) genes against rice blast disease. Phytopathology. 107, 711–720. Supplementary material associated with this article can be found, in Srivastava, D., Shamim, M., Kumar, M., Mishra, A., Pandey, P., Kumar, D., Yadav, P., the online version, at doi:10.1016/j.stress.2024.100465. Siddiqui, M.H., Singh, K.N., 2017. Current status of conventional and molecular interventions for blast resistance in rice. Rice Sci. 24, 299–321. 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