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Key Message: Rice CC-BED NLR Xa1 recognizes TAL effectors through the interaction between ERF101 and TAL effectors. The rice Xa1 gene encodes a nucleotide-binding leucine-rich repeat receptor with an N-terminal coiled coil-zinc finger BED (CC-BED) domain. Xa1 recognizes the transcription activator-like (TAL) effectors of Xanthomonas oryzae pv. oryzae (Xoo) in the nucleus, triggering a number of immune responses, including hypersensitive cell death. We previously discovered that the rice transcription factor ERF101 directly interacts with Xa1, and functions as a positive regulator of Xa1-dependent immunity. However, the involvement of ERF101 in Xa1-induced immunity remains unclear. We herein demonstrated that the expression of the CC-BED domain in rice protoplasts inhibited Xa1-induced cell death. However, the CC-BED C165A,C168A domain which has mutations of cysteine residues conserved in the zinc-finger motifs of BED domains and is essential for forming tetrahedral coordination geometry, failed to inhibit cell death or interact with ERF101. Therefore, Xa1-induced cell death appears to depend on the interaction between the BED domain and ERF101. In addition, we generated transgenic plants overexpressing N-terminal or C-terminal FLAG-tagged ERF101. FLAG-ERF101 transgenic plants exhibited reduced levels of Xa1-mediated immunity against Xoo, even though the overexpression of ERF101-FLAG or non-tagged ERF101 enhanced immunity. This result was consistent with the CC-BED domain interacting with C-terminal tagged ERF101, but not N-terminal tagged ERF101, whereas N-terminal and C-terminal tagged ERF101 both interacted with TAL effectors. Therefore, the interaction between the BED domain and ERF101 appears to be essential for the recognition of TAL effectors by Xa1., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no conflict of interest., (© 2025. The Author(s).)

Key Message: OsMYB1 negatively mediates rice resistance to brown planthopper and rice blight. Additionally, OsMYB1 interacts with OsSPL14 and antagonizes its function by oppositely regulating downstream resistance-related genes. In their natural habitats, plants are concurrently attacked by different biotic factors. Xanthomonas oryzae pv. oryzae (Xoo) is a pathogen that severely deteriorates rice yield and quality, and brown planthopper (BPH; Nilaparvata lugens) is a rice specific insect pest with the damage topping other pathogens. Although genes for respective resistance to BPH and Xoo have been widely reported, few studies pay attention to simultaneous resistance to both. In this study, we identified a MYB transcription factor, OsMYB1, which exhibited diverse transcriptional regulatory capabilities and a negative regulatory role in resistance to both BPH and Xoo. Biochemical and genetic analysis proved OsMYB1 to be a TF that could interact with OsSPL14, a positive regulator of rice resistance to Xoo. OsSPL14 mutants showed increased sensitivity to BPH, suggesting that OsSPL14 is contrary to OsMYB1 in regulating rice resistance to these two biotic stresses. Consistently, OsMYB1 and OsSPL14 displayed opposite functions in regulating defense-related genes. OsMYB1 can form transcription regulation complexes with repressor OsJAZs instead of co-repressor TOPLESS to possibly realize its transcriptional repression function. Taken together, we concluded that two interacting TFs in rice, OsMYB1 and OsSPL14, played antagonistic roles in regulating resistance to BPH and Xoo., Competing Interests: Declarations. Conflict of interest: The authors declare no conflict of interest., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: By deploying a multi-omics approach, we unraveled the mechanisms that might help rice to combat Yellow Stem Borer infestation, thus providing insights and scope for developing YSB resistant rice varieties. Yellow Stem Borer (YSB), Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae), is a major pest of rice, that can lead to 20-60% loss in rice production. Effective management of YSB infestation is challenged by the non-availability of adequate sources of resistance and poor understanding of resistance mechanisms, thus necessitating studies for generating resources to breed YSB resistant rice and to understand rice-YSB interaction. In this study, by using bulk-segregant analysis in combination with next-generation sequencing, Quantitative Trait Loci (QTL) intervals in five rice chromosomes were mapped that could be associated with YSB resistance at the vegetative phase in a resistant rice line named SM92. Further, multiple SNP markers that showed significant association with YSB resistance in rice chromosomes 1, 5, 10, and 12 were developed. RNA-sequencing of the susceptible and resistant lines revealed several genes present in the candidate QTL intervals to be differentially regulated upon YSB infestation. Comparative transcriptome analysis revealed a putative candidate gene that was predicted to encode an alpha-amylase inhibitor. Analysis of the transcriptome and metabolite profiles further revealed a possible link between phenylpropanoid metabolism and YSB resistance. Taken together, our study provides deeper insights into rice-YSB interaction and enhances the understanding of YSB resistance mechanism. Importantly, a promising breeding line and markers for YSB resistance have been developed that can potentially aid in marker-assisted breeding of YSB resistance among elite rice cultivars., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: Pi65, a leucine-rich repeat receptor-like kinase (LRR-RLK) domain cloned from Oryza sativa japonica, is a novel rice blast disease resistance gene. Rice blast seriously threatens rice production worldwide. Utilizing the rice blast resistance gene to breed rice blast-resistant varieties is one of the best ways to control rice blast disease. Using a map-based cloning strategy, we cloned a novel rice blast resistance gene, Pi65, from the resistant variety GangYu129 (abbreviated GY129, Oryza sativa japonica). Overexpression of Pi65 in the susceptible variety LiaoXing1 (abbreviated LX1, Oryza sativa japonica) enhanced rice blast resistance, while knockout of Pi65 in GY129 resulted in susceptibility to rice blast disease. Pi65 encodes two transmembrane domains, with 15 LRR domains and one serine/threonine protein kinase catalytic domain, conferring resistance to isolates of Magnaporthe oryzae (abbreviated M. oryzae) collected from Northeast China. There were sixteen amino acid differences between the Pi65 resistance and susceptible alleles. Compared with the Pi65-resistant allele, the susceptible allele exhibited one LRR domain deletion. Pi65 was constitutively expressed in whole plants, and it could be induced in the early stage of M. oryzae infection. Transcriptome analysis revealed that numerous genes associated with disease resistance were specifically upregulated in GY129 24 h post inoculation (HPI); in contrast, photosynthesis and carbohydrate metabolism-related genes were particularly downregulated at 24 HPI, demonstrating that disease resistance-associated genes were activated in GY129 (carrying Pi65) after rice blast fungal infection and that cellular basal metabolism and energy metabolism were inhibited simultaneously. Our study provides genetic resources for improving rice blast resistance and enriches the study of rice blast resistance mechanisms., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The bacterial leaf blight in rice caused by Xanthomonas oryzae pv. oryzae (Xoo) affects crop losses worldwide. In spite of developing resistant varieties by introgressing different Xa genes, the occurrence of diseases is evident. Here we report identification of several genes that are associated with improved plant immunity against Xoo in a resistant genotype BPT-5204 in comparison with susceptible genotype TN-1. The RNA sequencing information was developed to identify the genes that could provide durable resistance in rice. Xoo-resistant rice genotype BPT-5204 with Xa 5, 13 and 21 genes is compared with sensitive Taichung Native 1 (TN-1) to identify the genetic pathways and gene networks involved in resistance mechanisms. The higher levels of salicylic acid resulted in upregulation of many pathogenesis-related (PR) and redox protein encoding transcripts which resulted in higher hypersensitive response in BPT-5204. Many Serine/threonine protein kinase, leucine-rich repeat (LRR) transmembrane protein kinase, protein kinase family genes, Wall-associated kinase (WAK) were upregulated that resulted in activation of bZIP, WRKY, MYB, DOF and HSFs transcription factors that are associated with improved plant immunity. The study provided roles of many genes and their associated plant immunity pathways that can be used for developing resistant rice cultivars., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Key Message: Transgenic rice overexpressing PLCP attenuated the virulence of Xanthomonas oryzae pv. oryzae through extensive activation of transduction signal and transcription activities that orchestrate downstream responses including the biosynthesis of secondary metabolites and up-regulation of several pathogenesis-related proteins. High-throughput transcriptome investigations of plant immunity highlight the complexity of gene networks leading to incompatible interaction with the pathogen. Accumulating findings implicate papain-like cysteine proteases (PLCPs) as a central hub in plant defense. While diverse roles of PLCPs in different pathosystems have become more evident, information on gene networks and signaling pathways necessary to orchestrate downstream responses are lacking. To understand the biological significance of cysteine protease against Xanthomonas oryzae pv. oryzae, PLCP overexpression and knockout rice lines were generated. The pathogenicity test revealed the attenuation of Xanthomonas oryzae pv. oryzae race K3a virulence in transgenic lines which is ascribed to high hydrogen peroxide and free salicylic acid accumulation. Next-generation sequencing of RNA from transgenic and wild-type plants identified 1597 combined differentially expressed genes, 1269 of which were exclusively regulated in the transgenic libraries. It was found that PLCP aids rice to circumvent infection through the extensive activation of transduction signal and transcription factors that orchestrate downstream responses, including up-regulation of multiple pathogenesis-related proteins and biosynthesis of secondary metabolites.

Assessment of temperature effect on plant resistance against diseases has become essential under climate change scenario as temperature rise is anticipated to modify host resistance. To determine temperature influence on resistance gene, a pair of near-isogenic rice lines differing for the Pi54 resistance gene was assessed against leaf blast. Blast resistance was determined as the extent of infection efficiency (IE) and sporulation (SP) at suboptimal (22 °C and 32 °C) and optimal temperature (27 °C) of pathogen aggressiveness. Relative resistance for IE and SP was higher at suboptimal temperature as compared to that of optimal temperature. Maximum level of resistance was at 22 °C where higher levels of expression of Pi54 and defence-regulatory transcription factor WRKY45 were also noted. At 32 °C, although some level of resistance noted, but level of Pi54 and WRKY45 expression was too low, suggesting that resistance recorded at higher temperature was due to reduced pathogen aggressiveness. At the optimal temperature for pathogen aggressiveness, comparatively lower levels of Pi54 and WRKY45 expression suggest possible temperature-induced interruption of the defence processes. The variation in resistance patterns modulated by temperature is appeared to be due to pathogen's sensitivity to temperature that leads to varying levels of Pi54 gene activation. Quick and violent activity of the pathogen at optimal temperature came into sight for the interruption of defence process activated by Pi54 gene. Evaluation of blast resistance genes under variable temperature conditions together with weather data could be applied in screening rice genotypes for selection of resistance having resilience to temperature rise.

Rhizoctonia solani causes rice sheath blight, an important disease affecting the growth of rice (Oryza sativa L.). Attempts to control the disease have met with little success. Based on transcriptional profiling, we previously identified more than 11,947 common differentially expressed genes (TPM > 10) between the rice genotypes TeQing and Lemont. In the current study, we extended these findings by focusing on an analysis of gene co-expression in response to R. solani AG1 IA and identified gene modules within the networks through weighted gene co-expression network analysis (WGCNA). We compared the different genes assigned to each module and the biological interpretations of gene co-expression networks at early and later modules in the two rice genotypes to reveal differential responses to AG1 IA. Our results show that different changes occurred in the two rice genotypes and that the modules in the two groups contain a number of candidate genes possibly involved in pathogenesis, such as the VQ protein. Furthermore, these gene co-expression networks provide comprehensive transcriptional information regarding gene expression in rice in response to AG1 IA. The co-expression networks derived from our data offer ideas for follow-up experimentation that will help advance our understanding of the translational regulation of rice gene expression changes in response to AG1 IA.

An alternative method to control rice blast (Magnaporthe oryzae) is to include biological agent in the disease management strategy. The objective of this study was to assess the leaf blast-suppressing effects of rice phylloplane fungi. One Cladosporium sp. phylloplane fungus was shown to possess biocontrolling traits based on its morphological characteristics and an analysis of its 18S ribosomal DNA. Experiments aimed at determining the optimal time to apply the bioagent and the mechanisms involved in its rice blast-suppressing activities were performed under controlled greenhouse conditions. We used foliar spraying to apply the Cladosporium sp. 48 h prior to applying the pathogen, and we found that this increased the enzymatic activity. Furthermore, in vitro tests performed using isolate C24 showed that it possessed the ability to secrete endoxylanases and endoglucanases. When Cladosporium sp. was applied either prior to or simultaneous with the pathogen, we observed a significant increase in defence enzyme activity, and rice blast was suppressed by 84.0 and 78.6 %, respectively. However, some enzymes showed higher activity at 24 h while others did so at 48 h after the challenge inoculation. Cladosporium sp. is a biological agent that is capable of suppressing rice leaf blast by activating biochemical defence mechanisms in rice plants. It is highly adapted to natural field conditions and should be included in further studies aimed at developing strategies to support ecologically sustainable disease management and reduce environmental pollution by the judicious use of fungicidal sprays.

Key Message: OsWRKY51 functions as a positive transcriptional regulator in defense signaling against Xanthomonas oryzae pv. oryzae by direct DNA binding to the promoter of defense related gene, OsPR10a. OsWRKY51 in rice (Oryza sativa L.) is induced by exogenous salicylic acid (SA) and inoculation with Xanthomonas oryzae pv. oryzae (Xoo). To examine the role of OsWRKY51 in the defense response of rice, we generated OsWRKY51 overexpressing and underexpressing transgenic rice plants. OsWRKY51-overexpressing transgenic rice lines were more resistant to Xoo and showed greater expression of defense-related genes than wild-type (WT) plants, while OsWRKY51-underexpressing lines were more susceptible to Xoo and showed less expression of defense-associated genes than WT plants. Transgenic lines overexpressing OsWRKY51 showed growth retardation compared to WT plants. In contrast, transgenic lines underexpressing OsWRKY51 by RNA interference showed similar plant height with WT plants. Transient expression of OsWRKY51-green fluorescent protein fusion protein in rice protoplasts revealed that OsWRKY51 was localized in the nucleus. OsWRKY51 bound to the W-box and WLE1 elements of the OsPR10a promoter. Based on these results, we suggest that OsWRKY51 is a positive transcriptional regulator of defense signaling and has direct DNA binding ability to the promoter of OsPR10a, although it is reported to be a negative regulator in GA signaling.

The Asian rice gall midge, Orseolia oryzae, is a serious insect pest causing extensive yield loss. Interaction between the gall midge and rice genotypes is known to be on a gene-for-gene basis. Here, we report molecular basis of HR- (hypersensitive reaction-negative) type of resistance in Aganni (an indica rice variety possessing gall midge resistance gene Gm8) through the construction and analysis of a suppressive subtraction hybridization (SSH) cDNA library. In all, 2,800 positive clones were sequenced and analyzed. The high-quality ESTs were assembled into 448 non-redundant gene sequences. Homology search with the NCBI databases, using BlastX and BlastN, revealed that 73% of the clones showed homology to genes with known function and majority of ESTs belonged to the gene ontology category 'biological process'. Validation of 27 putative candidate gall midge resistance genes through real-time PCR, following gall midge infestation, in contrasting parents and their derived pre-NILs (near isogenic lines) revealed induction of specific genes related to defense and metabolism. Interestingly, four genes, belonging to families of leucine-rich repeat (LRR), heat shock protein (HSP), pathogenesis related protein (PR), and NAC domain-containing protein, implicated in conferring HR+ type of resistance, were found to be up-regulated in Aganni. Two of the reactive oxygen intermediates (ROI)-scavenging-enzyme-coding genes Cytosolic Ascorbate Peroxidase1, 2 (OsAPx1 and OsAPx2) were found up-regulated in Aganni in incompatible interaction possibly suppressing HR. We suggest that Aganni has a deviant form of inducible, salicylic acid (SA)-mediated resistance but without HR.

Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, secretes several cell wall degrading enzymes including cellulase (ClsA) and lipase/esterase (LipA). Prior treatment of rice leaves with purified cell wall degrading enzymes such as LipA can confer enhanced resistance against subsequent X. oryzae pv. oryzae infection. To understand LipA-induced rice defense responses, microarray analysis was performed 12 h after enzyme treatment of rice leaves. This reveals that 867 (720 upregulated and 147 downregulated) genes are differentially regulated (≥2-fold). A number of genes involved in defense, stress, signal transduction, and catabolic processes were upregulated while a number of genes involved in photosynthesis and anabolic processes were downregulated. The microarray data also suggested upregulation of jasmonic acid (JA) biosynthetic and JA-responsive genes. Estimation of various phytohormones in LipA-treated rice leaves demonstrated a significant increase in the level of JA-Ile (a known active form of JA) while the levels of other phytohormones were not changed significantly with respect to buffer-treated control. This suggests a role for JA-Ile in cell wall damage induced innate immunity. Furthermore, a comparative analysis of ClsA- and LipA-induced rice genes has identified key rice functions that might be involved in elaboration of damage-associated molecular pattern (DAMP)-induced innate immunity.

Key Message: Our studies indicate a potential important elicitor candidate which can aid in the fight against a worldwide disease, rice blast. In this study, we report the purification, identification, characterization, and gene cloning of a novel hypersensitive response-inducing protein elicitor (MoHrip2) secreted from an important pathogenic fungus, Magnaporthe oryzae. The protein fraction was isolated from the culture filtrate of M. oryzae and identified by de novo sequencing. The elicitor-encoding gene mohrip2 was cloned following sequence comparison and PCR amplification. This 459-bp gene encodes a 152-residue polypeptide that contains an 18-residue signal peptide and exhibits a pI of 4.72 and an apparent molecular mass of 16 kDa. The hypothetical protein, MoHrip2, was expressed in Escherichia coli, and both the recombinant and the endogenous protein caused necrotic lesions in tobacco leaves. In addition to phenolic compound deposition and alkalization of the extracellular medium, MoHrip2 also induced hydrogen peroxide production and nitric oxide accumulation in tobacco cells. Moreover, rice seedlings treated with MoHrip2 exhibited pronounced resistance to M. oryzae compared with control seedlings.

Key Message: Expression levels of rice VQ motif-containing genes in response to pathogen infection vary among pathogens, and some of the genes are co-expressed with defense-response WRKY genes. Recent studies have revealed that some VQ (FxxxVQxLTG) motif-containing proteins in plants partner with WRKY transcription factors to participate in their functions. Accumulating information suggests that WRKY proteins play important roles in the response of rice plants to pathogen infection. However, the functions of rice VQ motif-containing proteins are unknown. To explore whether VQ motif-containing proteins are involved in defense against pathogens in rice, we performed a comprehensive expression analysis of the genes for these proteins. The rice VQ motif-containing family consists of 40 genes, all of which encode proteins harboring a 21-amino acid VQ-containing motif, which in turn contains the known VQ motif. On the basis of their phylogenetic relationships and tissue-specific and developmental stage-specific expression characteristics, we transcriptionally analyzed 13 representative genes in rice responses to three pathogens: Xanthomonas oryzae pv. oryzae, which causes bacterial blight disease; X. oryzae pv. oryzicola, which causes bacterial streak disease; and Magnaporthe oryzae, which causes fungal blast disease. The expression of some of the genes changed markedly in response to infection by at least one of the pathogen species, and some of the genes also showed markedly different expression in resistant and susceptible reactions. In addition, some defense-responsive VQ motif-containing genes were co-expressed with defense-response WRKY genes. These results provide a new perspective on the putative roles of rice VQ motif-containing proteins and their putative WRKY partners in rice-pathogen interactions.

Key Message: OsMPK3 is a TEY-type rice MAPK belonging to Group C and directly phosphorylates OsbHLH65 in the nucleus. OsMPK3 and OsbHLH65 are induced by biotic stress and defense-related hormones. Mitogen-activated protein kinases (MAPKs) are involved in the majority of signaling pathways that regulate plant development and stress tolerance via the phosphorylation of target molecules. Plant MAPKs are classified into two subtypes, TEY and TDY, according to the TxY (x = E or D) motif in their activation loop, and the TDY motif is unique to plant MAPKs. In rice, 17 MAPKs have been classified into six groups. To date, the functions of many TDY-type rice MAPKs have been characterized, but little is known of the TEY-type MAPKs in Group C and their possible target substrates. In the study reported here, we determined that a TEY-type rice MAPK belonging to subgroup C, named OsMPK3, phosphorylates its substrate OsbHLH65 in the nucleus. Our electrophoresis mobility shift assay results revealed that OsbHLH65 specifically binds to the E-box cis-element, but not to the G-box. Both OsMPK3 and OsbHLH65 were induced by treatments with rice blast (Magnaporthe grisea), brown planthopper (Nilaparvata lugens), and defense-related hormones, such as methyl jasmonic acid and salicylic acid. Our results suggest the possibility that OsMPK3 contributes to the defense signal transduction by phosphorylating the basic helix-loop-helix transcription factor.

Magnaporthe oryzae causes rice blast that is one of the most devastating diseases of rice worldwide. Highly variable nature of this fungus has evolved itself against major resistance genes in newly released rice varieties. Understanding the population structure of this fungus is essential for proper utilization of the rice blast resistance genes in rice crop plants. In the present study, we analyzed 133 isolates of M. oryzae from ten countries to find the allelic variation of Avr-Pita gene that is triggering Pita-mediated resistance in rice plant. The diversity analysis of these alleles showed higher level of nucleotide variation in the coding regions than the noncoding regions. Evolutionary analysis of these alleles indicates that Avr-Pita gene is under purifying selection to favor its major alleles in 133 isolates analyzed in this study. We hypothesize that the selection of favorable Avr-Pita allele in these isolates may occur through a genetic mechanism known as recurrent selective sweeps. A total of 22 functional Avr-Pita protein variants were identified in this study. Insertion of Pot3 transposable element into the promoter of Avr-Pita gene was identified in virulent isolates and was suggested that mobility of repeat elements in avirulence genes of M. oryzae seems to help in emergence of new virulent types of the pathogen. Allele-specific markers developed in this study will be helpful to identify a particular type of Avr-Pita allele from M. oryzae population which can form the basis for the deployment of Pita gene in different epidemiological regions.

Rice sheath blight and blast caused by Rhizoctonia solani Kühn and Magnorpathe oryzae respectively, are the two most destructive fungal diseases in rice. With no genetic natural traits conferring resistance to sheath blight, transgenic manipulation provides an obvious approach. In this study, the rice basic chitinase gene (RCH10) and the alfalfa β-1,3-glucanase gene (AGLU1) were tandemly inserted into transformation vector pBI101 under the control of 35S promoter with its enhancer sequence to generate a double-defense gene expression cassette pZ100. The pZ100 cassette was transformed into rice (cv. Taipei 309) by Agrobacterium-mediated transformation. More than 160 independent transformants were obtained and confirmed by PCR. Northern analysis of inheritable progenies revealed similar levels of both RCH10 and AGLU1 transcripts in the same individuals. Disease resistance to both sheath blight and blast was challenged in open field inoculation. Immunogold detection revealed that RCH10 and AGLU1 proteins were initially located mainly in the chloroplasts and were delivered to the vacuole and cell wall upon infection, suggesting that these subcellular compartments act as the gathering and execution site for these anti-fungal proteins. We also observed that transgenic seeds display lower germination rate and seedling vigor, indicating that defense enhancement might be achieved at the expense of development.

The plant hormone jasmonate (JA) fulfils essential roles in plant defense and development. While most of our current understanding of the JA pathway comes from the dicotyledonous model plant Arabidopsis thaliana, new studies in monocotyledonous plants are providing additional insights into this important hormone signaling pathway. In this review, we present a comparative overview of the JA biosynthetic and signaling pathways in monocots. We highlight recent studies that have revealed molecular mechanisms (mostly conserved but also diverged) underlying JA signaling and biosynthesis in the economically important plants: maize and rice. A better understanding of the JA pathway in monocots should lead to significant improvements in pest and pathogen resistance in cereal crops, which provide the bulk of the world's food and feed supply.

Sheath blight (SB), caused by Rhizoctonia solani kühn, is one of the most serious global rice diseases. No major resistance genes to SB have been identified so far. All discovered loci are quantitative resistance to rice SB. The qSB-11(LE) resistance quantitative trait locus (QTL) has been previously reported on chromosome 11 of Lemont (LE). In this study, we report the precise location of qSB-11 (LE) . We developed a near isogenic line, NIL-qSB11(TQ), by marker-assisted selection that contains susceptible allele(s) from Teqing (TQ) at the qSB-11 locus in the LE genetic background. NIL-qSB11(TQ) shows higher susceptibility to SB than LE in both field and greenhouse tests, suggesting that this region of LE contains a QTL contributing to SB resistance. In order to eliminate the genetic background effects and increase the accuracy of phenotypic evaluation, a total of 112 chromosome segment substitution lines (CSSLs) with the substituted segment specific to the qSB-11 (LE) region were produced as the fine mapping population. The genetic backgrounds and morphological characteristics of these CSSLs are similar to those of the recurrent parent LE. The donor TQ chromosomal segments in these CSSL lines contiguously overlap to bridge the qSB-11 (LE) region. Through artificial inoculation, all CSSLs were evaluated for resistance to SB in the field in 2005. For the recombinant lines, their phenotypes were evaluated in the field for another 3 years and during the final year were also evaluated in a controlled greenhouse environment, showing a consistent phenotype in SB resistance across years and conditions. After comparing the genotypic profile of each CSSL with its phenotype, we are able to localize qSB-11 (LE) to the region defined by two cleaved-amplified polymorphic sequence markers, Z22-27C and Z23-33C covering 78.871 kb, based on the rice reference genome. Eleven putative genes were annotated within this region and three of them were considered the most likely candidates. The results of this study will greatly facilitate the cloning of the genes responsible for qSB-11 (LE) and marker-assisted breeding to incorporate qSB-11 (LE) into other rice cultivars.

Spontaneous sequence changes and the selection of beneficial mutations are driving forces of gene diversification and key factors of evolution. In highly dynamic co-evolutionary processes such as plant-pathogen interactions, the plant's ability to rapidly adapt to newly emerging pathogens is paramount. The hexaploid wheat gene Lr34, which encodes an ATP-binding cassette (ABC) transporter, confers durable field resistance against four fungal diseases. Despite its extensive use in breeding and agriculture, no increase in virulence towards Lr34 has been described over the last century. The wheat genepool contains two predominant Lr34 alleles of which only one confers disease resistance. The two alleles, located on chromosome 7DS, differ by only two exon-polymorphisms. Putatively functional homoeologs and orthologs of Lr34 are found on the B-genome of wheat and in rice and sorghum, but not in maize, barley and Brachypodium. In this study we present a detailed haplotype analysis of homoeologous and orthologous Lr34 genes in genetically and geographically diverse selections of wheat, rice and sorghum accessions. We found that the resistant Lr34 haplotype is unique to the wheat D-genome and is not found in the B-genome of wheat or in rice and sorghum. Furthermore, we only found the susceptible Lr34 allele in a set of 252 Ae. tauschii genotypes, the progenitor of the wheat D-genome. These data provide compelling evidence that the Lr34 multi-pathogen resistance is the result of recent gene diversification occurring after the formation of hexaploid wheat about 8,000 years ago.

Glycoside hydrolase family 19 chitinases (EC 3.2.1.14) widely distributed in plants, bacteria and viruses catalyse the hydrolysis of chitin and play a major role in plant defense mechanisms and development. Rice possesses several classes of chitinase, out of which a single structure of class I has been reported in PDB to date. In the present study an attempt was made to gain more insight into the structure, function and evolution of class I, II and IV chitinases of GH family 19 from rice. The three-dimensional structures of chitinases were modelled and validated based on available X-ray crystal structures. The structural study revealed that they are highly α-helical and bilobed in nature. These enzymes are single or multi domain and multi-functional in which chitin-binding domain (CBD) and catalytic domain (CatD) are present in class I and IV whereas class II lacks CBD. The CatD possesses a catalytic triad which is thought to be involved in catalytic process. Loop III, which is common in all three classes of chitinases, reflects that it may play a significant role in their function. Our study also confirms that the absence and presence of different loops in GH family 19 of rice may be responsible for various sized products. Molecular phylogeny revealed chitinases in monocotyledons and dicotyledons differed from each other forming two different clusters and may have evolved differentially. More structural study of this enzyme from different plants is required to enhance the knowledge of catalytic mechanism and substrate binding.

To increase immune responses of plant-based vaccines in intestine mucosal immune systems, a synthetic neutralizing epitope (sCOE) gene of porcine epidemic diarrhea virus (PEDV) was fused with M cell-targeting ligand (Co1) and introduced into a plant expression vector under the control of rice amylase 3D promoter. The sCOE-Co1 fusion gene was introduced into rice calli via the particle bombardment-mediated transformation method. The stable integration and transcriptional expression of the sCOE-Co1 fusion gene was confirmed by genomic DNA PCR amplification and Northern blot analysis, respectively. The expression of the COE-Co1 fusion protein was confirmed by immunoblot analysis. The highest expression level of the COE-Co1 fusion protein reached 0.083 % of the total soluble protein according to quantitative densitometry of Western blot analysis. Mice immunized with transgenic rice calli protein extracts induced significant serum IgG and fecal IgA antibody levels against purified bacterial COE. The systemic and mucosal immune responses were confirmed by measuring COE-specific IgG and IgA antibody-secreting cells in the lymphocytes extracted from the spleen and COE-specific IgA antibody-secreting cells in the Peyer's patches from immunized mice. These results indicated that oral immunization of plant-produced COE-Co1 fusion protein could elicit efficient systemic and mucosal immune responses against the COE antigen. Key message Neutralizing epitope from porcine epidemic diarrhea virus-M cell targeting ligand fusion protein was produced in transgenic rice calli and elicited systemic and mucosal immune responses by oral administration in mice.

Rice stripe disease, caused by rice stripe virus (RSV) is a serious constraint to rice production in subtropical regions of East Asia. We performed fine mapping of a RSV resistance QTL on chromosome 11, qSTV11 ( SG ), using near-isogenic lines (NILs, BC(6)F(4)) derived from a cross between the highly resistant variety, Shingwang, and the highly susceptible variety, Ilpum, using 11 insertion and deletion (InDel) markers. qSTV11 ( SG ) was localized to a 150-kb region between InDel 11 (17.86 Mbp) and InDel 5 (18.01 Mbp). Among the two markers in this region, InDel 7 is diagnostic of RSV resistance in 55 Korean japonica and indica rice varieties. InDel 7 could also distinguish the allele type of Nagdong, Shingwang, Mudgo, and Pe-bi-hun from Zenith harboring the Stv-b ( i ) allele. As a result, qSTV11 ( SG ) is likely to be the Stv-b ( i ) allele. There were 21 genes in the 150-kb region harboring the qSTV11 ( SG ) locus. Three of these genes, LOC_Os11g31430, LOC_Os11g31450, and LOC_Os11g31470, were exclusively expressed in the susceptible variety. These expression profiles were consistent with the quantitative nature along with incomplete dominance of RSV resistance. Sequencing of these genes showed that there were several amino acid substitutions between susceptible and resistant varieties. Putative functions of these candidate genes for qSTV11 (SG) are discussed.

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

Quantitative trait loci (QTLs) for resistance to rice blast offer a potential source of durable disease resistance in rice. However, few QTLs have been validated in progeny testing, on account of their small phenotypic effects. To understand the genetic basis for QTL-mediated resistance to blast, we dissected a resistance QTL, qBR4-2, using advanced backcross progeny derived from a chromosome segment substitution line in which a 30- to 34-Mb region of chromosome 4 from the resistant cultivar Owarihatamochi was substituted into the genetic background of the highly susceptible Aichiasahi. The analysis resolved qBR4-2 into three loci, designated qBR4-2a, qBR4-2b, and qBR4-2c. The sequences of qBR4-2a and qBR4-2b, which lie 181 kb apart from each other and measure, 113 and 32 kb, respectively, appear to encode proteins with a putative nucleotide-binding site (NBS) and leucine-rich repeats (LRRs). Sequence analysis of the donor allele of qBR4-2a, the region with the largest effect among the three, revealed sequence variations in the NBS-LRR region. The effect of qBR4-2c was smallest among the three, but its combination with the donor alleles of qBR4-2a and qBR4-2b significantly enhanced blast resistance. qBR4-2 comprises three tightly linked QTLs that control blast resistance in a complex manner, and thus gene pyramiding or haplotype selection is the recommended strategy for improving QTL-mediated resistance to blast disease through the use of this chromosomal region.

The dominant rice blast resistance gene, Pi54 confers resistance to Magnaporthe oryzae in different parts of India. In our effort to identify more effective forms of this gene, we isolated an orthologue of Pi54 named as Pi54rh from the blast-resistant wild species of rice, Oryza rhizomatis, using allele mining approach and validated by complementation. The Pi54rh belongs to CC-NBS-LRR family of disease resistance genes with a unique Zinc finger (C(3)H type) domain. The 1,447 bp Pi54rh transcript comprises of 101 bp 5'-UTR, 1,083 bp coding region and 263 bp 3'-UTR, driven by pathogen inducible promoter. We showed the extracellular localization of Pi54rh protein and the presence of glycosylation, myristoylation and phosphorylation sites which implicates its role in signal transduction process. This is in contrast to other blast resistance genes that are predicted to be intracellular NBS-LRR-type resistance proteins. The Pi54rh was found to express constitutively at basal level in the leaves, but upregulates 3.8-fold at 96 h post-inoculation with the pathogen. Functional validation of cloned Pi54rh gene using complementation test showed high degree of resistance to seven isolates of M. oryzae collected from different geographical locations of India. In this study, for the first time, we demonstrated that a rice blast resistance gene Pi54rh cloned from wild species of rice provides broad spectrum resistance to M. oryzae hence can be used in rice improvement breeding programme.

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.

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is an important foliar disease of wheat worldwide. Wild emmer (Triticum turgidum var. dicoccoides) is a valuable genetic resource for improving disease resistance in common wheat. A powdery mildew resistance gene conferring resistance to B. graminis f. sp. tritici isolate E09 at the seedling and adult stages was identified in wild emmer accession IW170 introduced from Israel. An incomplete dominant gene, temporarily designated MlIW170, was responsible for the resistance. Through molecular marker and bulked segregant analyses of an F(2) population and F(3) families derived from a cross between susceptible durum wheat line 81086A and IW170, MlIW170 was located in the distal chromosome bin 2BS3-0.84-1.00 and flanked by SSR markers Xcfd238 and Xwmc243. MlIW170 co-segregated with Xcau516, an STS marker developed from RFLP marker Xwg516 that co-segregated with powdery mildew resistance gene Pm26 on 2BS. Four EST-STS markers, BE498358, BF201235, BQ160080, and BF146221, were integrated into the genetic linkage map of MlIW170. Three AFLP markers, XPaacMcac, XPagcMcta, XPaacMcag, and seven AFLP-derived SCAR markers, XcauG2, XcauG3, XcauG6, XcauG8, XcauG10, XcauG20, and XcauG25, were linked to MlIW170. XcauG3, a resistance gene analog (RGA)-like sequence, co-segregated with MlIW170. The non-glaucousness locus Iw1 was 18.77 cM distal to MlIW170. By comparative genomics of wheat-Brachypodium-rice genomic co-linearity, four EST-STS markers, CJ658408, CJ945509, BQ169830, CJ945085, and one STS marker XP2430, were developed and MlIW170 was mapped in an 2.69 cM interval that is co-linear with a 131 kb genomic region in Brachypodium and a 105 kb genomic region in rice. Four RGA-like sequences annotated in the orthologous Brachypodium genomic region could serve as chromosome landing target regions for map-based cloning of MlIW170.

Rice (Oryza sativa) cv. Nipponbare expresses non-host resistance (NHR) to the wheat leaf rust fungus, Puccinia triticina f. sp. tritici (Ptt). When the leaves of cv. Nipponbare were inoculated with Ptt, approx 93% of the urediniospores germinated on the leaf surface, but only 10% of the germinated spores formed appressoria over the stomata at one day post inoculation (1 dpi). Hydrogen peroxide (H(2)O(2)) accumulated in host cells around the appressoria at 3 dpi. Approx. 3% of the appressoria produced short hyphae inside the leaf, and fluorescence was observed in tissue invaded by the hyphae by 7 dpi. At 22 dpi, 0.2% of the sites with appressoria formed branching infection hypha in mesophyll cells, but no substomatal vesicles, haustorial mother cells or haustoria were observed. Proteins were extracted from leaves 3 dpi and analyzed by two-dimensional gel electrophoresis (2-DE). A total 33 spots were reproducibly up-regulated and 9 were down-regulated by infection compared to the water inoculated control. Of these, 30 were identified by MALDI-TOF Mass Spectrometry. The identified proteins participate in defense/stress responses, energy/carbohydrate metabolism, oxidation-reduction processes, protein folding/turnover/cleavage/degradation, signal transduction and cell death regulation. The results indicates that NHR of rice to Ptt is consistent with a shift in protein and energy metabolism, increased antimicrobial activities, possibly including phytoalexin accumulation and cell wall reinforcement, increased cell repair, antioxidive and detoxification reactions, and enhanced prevention of plant cell death. Nearly half of the up-regulated identified proteins were associated with chloroplast and mitochondrial physiology suggesting important roles for these organelles during NHR., (© Springer-Verlag 2011)

Plant fungal pathogens change their cell wall components during the infection process to avoid degradation by host lytic enzymes, and conversion of the cell wall chitin to chitosan is likely to be one infection strategy of pathogens. Thus, introduction of chitosan-degradation activity into plants is expected to improve fungal disease resistance. Chitosanase has been found in bacteria and fungi, but not in higher plants. Here, we demonstrate that chitosanase, Cho1, from Bacillus circulans MH-K1 has antifungal activity against the rice blast fungus Magnaporthe oryzae. Introduction of the cho1 gene conferred chitosanase activity to rice cells. Transgenic rice plants expressing Cho1 designed to be localized in the apoplast showed increased resistance to M. oryzae accompanied by increased generation of hydrogen peroxide in the infected epidermal cells. These results strongly suggest that chitosan exists in the enzyme-accessible surface of M. oryzae during the infection process and that the enhancement of disease resistance is attributable to the antifungal activity of the secreted Cho1 and to increased elicitation of the host defense response., (© Springer-Verlag 2011)

Rice chitinase (chi11) and tobacco osmotin (ap24) genes, which cause disruption of fungal cell wall and cell membrane, respectively, were stacked in transgenic rice to develop resistance against the sheath blight disease. The homozygous marker-free transgenic rice line CoT23 which harboured the rice chi11 transgene was sequentially re-transformed with a second transgene ap24 by co-transformation using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector pGV2260::pSSJ1 and a multi-copy binary vector pBin19∆nptII-ap24 in the same cell. pGV2260::pSSJ1 T-DNA carried the hygromycin phosphotransferase (hph) and β-glucuronidase (gus) genes. pBin19∆nptII-ap24 T-DNA harboured the tobacco osmotin (ap24) gene. Co-transformation of the gene of interest (ap24) with the selectable marker gene (SMG, hph) occurred in 12 out of 18 T(0) plants (67%). Segregation of hph from ap24 was accomplished in the T(1) generation in one (line 11) of the four analysed co-transformed plants. The presence of ap24 and chi11 transgenes and the absence of the hph gene in the SMG-eliminated T(1) plants of the line 11 were confirmed by DNA blot analyses. The SMG-free transgenic plants of the line 11 harboured a single copy of the ap24 gene. Homozygous, SMG-free T(2) plants of the transgenic line 11 harboured stacked transgenes, chi11 and ap24. Northern blot analysis of the SMG-free plants revealed constitutive expression of chi11 and ap24. The transgenic plants with stacked transgenes displayed high levels of resistance against Rhizoctonia solani. Thus, we demonstrate the development of transgene-stacked and marker-free transgenic rice by sequential Agrobacterium-mediated co-transformation with the same SMG.

A new bacterial blight recessive resistance gene xa34(t) was identified from the descendant of somatic hybridization between an aus rice cultivar (cv.) BG1222 and susceptible cv. IR24 against Chinese race V (isolate 5226). The isolate was used to test the resistance or susceptibility of F(1) progenies and reciprocal crosses of the parents. The results showed that F(1) progenies appeared susceptibility there were 128R (resistant):378S (susceptible) and 119R:375S plants in F(2) populations derived from two crosses of BG1222/IR24 and IR24/BG1222, respectively, which both calculates into a 1R:3S ratio. 320 pairs of stochastically selected SSR primers were used for genes' initial mapping. The screened results showed that two SSR markers, RM493 and RM446, found on rice chromosome 1 linked to xa34(t). Linkage analysis showed that these two markers were on both sides of xa34(t) with the genetic distances 4.29 and 3.05 cM, respectively. The other 50 SSR markers in this region were used for genes' fine mapping. The further results indicated that xa34(t) was mapped to a 1.42 cM genetic region between RM10927 and RM10591. In order to further narrow down the genomic region of xa34(t), 43 of insertion/deletion (Indel) markers (BGID1-43) were designed according to the sequences comparison between japonica and indica rice. Parents' polymorphic detection and linkage assay showed that the Indel marker BGID25 came closer to the target gene with a 0.4 cM genetic distance. A contig map corresponding to the locus was constructed based on the reference sequences aligned by the xa34(t) linked markers. Consequently, the locus of xa34(t) was defined to a 204 kb interval flanked by markers RM10929 and BGID25.

The indica rice cultivar, Teqing, shows a high level of resistance to rice stripe virus (RSV). It is believed that this resistance is controlled by the gene, qSTV11(TQ). For positional cloning of the resistance gene, a set of chromosome single segment substitution lines (CSSSLs) was constructed, all of which had the genetic background of the susceptible japonica cultivar, Lemont, with different single substituted segments of Teqing on chromosome 11. By identifying the resistance of the CSSSLs-2006 in a field within a heavily diseased area, the resistance gene qSTV11(TQ) was mapped between the markers Indel7 and RM229. Furthermore, in that region, six new markers were developed and 52 subregion CSSSLs (CSSSLs-2007) were constructed. The natural infection experiment was conducted again at different sites, with two replicates used in each site in order to identify the resistance phenotypes of the CSSSLs-2007 and resistant/susceptible controls in 2007. Through the results of 2007, qSTV11(TQ) was localized in a region defined by the markers, CAPs1 and Indel4. In order to further confirm the position of qSTV11(TQ), another set of subregion CSSSLs (CSSSLs-2009) was constructed. Finally, qSTV11(TQ) was localized to a 55.7 kb region containing nine annotated genes according to the genome sequence of japonica Nipponbare. The relationship between qSTV11(TQ) and Stvb-i (Hayano-Saito et al. in Theor Appl Genet 101:59-63, 2000) and the reliability of the markers used on both sides of qSTV11(TQ) for marker-assisted breeding of resistance to rice stripe disease are discussed.

The blast resistance gene Pik-p, mapping to the Pik locus on the long arm of rice chromosome 11, was isolated by map-based in silico cloning. Four NBS-LRR genes are present in the target region of cv. Nipponbare, and a presence/absence analysis in the Pik-p carrier cv. K60 excluded two of these as candidates for Pik-p. The other two candidates (KP3 and KP4) were expressed in cv. K60. A loss-of-function experiment by RNAi showed that both KP3 and KP4 are required for Pik-p function, while a gain-of-function experiment by complementation test revealed that neither KP3 nor KP4 on their own can impart resistance, but that resistance was expressed when both were introduced simultaneously. Both Pikp-1 (KP3) and Pikp-2 (KP4) encode coiled-coil NBS-LRR proteins and share, respectively, 95 and 99% peptide identity with the two alleles, Pikm1-TS and Pikm2-TS. The Pikp-1 and Pikp-2 sequences share only limited homology. Their sequence allowed Pik-p to be distinguished from Pik, Pik-s, Pik-m and Pik-h. Both Pikp-1 and Pikp-2 were constitutively expressed in cv. K60 and only marginally induced by blast infection.

We evaluated a large collection of Tos17 mutant panel lines for their reaction to three different races of Magnaporthe oryzae and identified a lesion mimic mutant, NF4050-8, that showed lesions similar to naturally occurring spl5 mutant and enhanced resistance to all the three blast races tested. Nested modified-AFLP using Tos17-specific primers and southern hybridization experiments of segregating individuals indicated that the lesion mimic phenotype in NF4050-8 is most likely due to a nucleotide change acquired during the culturing process and not due to Tos17 insertion per se. Inheritance and genetic analyses in two japonica × indica populations identified an overlapping genomic region of 13 cM on short arm of chromosome 7 that was linked with the lesion mimic phenotype. High-resolution genetic mapping using 950 F(3) and 3,821 F(4) plants of NF4050-8 × CO39 delimited a 35 kb region flanked by NBARC1 (5.262 Mb) and RM8262 (5.297 Mb), which contained 6 ORFs; 3 of them were 'resistance gene related' with typical NBS-LRR signatures. One of them harbored a NB-ARC domain, which had been previously demonstrated to be associated with cell death in animals. Microarray analysis of NF4050-8 revealed significant up-regulation of numerous defense/pathogenesis-related genes and down-regulation of heme peroxidase genes. Real-time PCR analysis of WRKY45 and PR1b genes suggested possible constitutive activation of a defense signaling pathway downstream of salicylic acid but independent of NH1 in these mutant lines of rice.

SHZ-2 is an indica rice cultivar that exhibits broad-spectrum resistance to rice blast; it is widely used as a resistance donor in breeding programs. To dissect the QTL responsible for broad-spectrum blast resistance, we crossed SHZ-2 to TXZ-13, a blast susceptible indica variety, to produce 244 BC(4)F(3) lines. These lines were evaluated for blast resistance in greenhouse and field conditions. Chromosomal introgressions from SHZ-2 into the TXZ-13 genome were identified using a single feature polymorphism microarray, SSR markers and gene-specific primers. Segregation analysis of the BC(4)F(3) population indicated that three regions on chromosomes 2, 6, and 9, designated as qBR2.1, qBR6.1, and qBR9.1, respectively, was associated with blast resistance and contributed 16.2, 14.9, and 22.3%, respectively, to the phenotypic variance of diseased leaf area (DLA). We further narrowed the three QTL regions using pairs of sister lines extracted from heterogeneous inbred families (HIF). Pairwise comparison of these lines enabled the determination of the relative contributions of individual QTL. The qBR9.1 conferred strong resistance, whereas qBR2.1 or qBR6.1 individually did not reduce disease under field conditions. However, when qBR2.1 and qBR6.1 were combined, they reduced disease by 19.5%, suggesting that small effect QTLs contribute to reduction of epidemics. The qBR6.1 and qBR9.1 regions contain nucleotide-binding sites and leucine rich repeats (NBS-LRR) sequences, whereas the qBR2.1 did not. In the qBR6.1 region, the patterns of expression of adjacent NBS-LRR genes were consistent in backcross generations and correlated with blast resistance, supporting the hypothesis that multiple resistance genes within a QTL region can contribute to non-race-specific quantitative resistance.

Brown planthopper (Nilaparvata lugens Stål, BPH) is one of the most destructive insect pests of rice. Exploring resistance genes from diverse germplasms and incorporating them into cultivated varieties are critical for controlling this insect. The rice variety Swarnalata was reported to carry a resistance gene (designated Bph6), which has not yet been assigned to a chromosome location and the resistance mechanism is still unknown. In this study, we identified and mapped this gene using the F(2) and backcrossing populations and characterized its resistance in indica 9311 and japonica Nipponbare using near isogenic lines (NILs). In analysis of 9311/Swarnalata F(2) population, the Bph6 gene was located on the long arm of chromosome 4 between the SSR markers RM6997 and RM5742. The gene was further mapped precisely to a 25-kb region delimited between the STS markers Y19 and Y9; and the distance between these markers is 25-kb in Nipponbare genome. The Bph6 explained 77.5% of the phenotypic variance of BPH resistance in F(2) population and 84.9% in BC(2)F(2) population. Allele from Swarnalata significantly increased resistance to the BPH, resulted in a reduced damage score. In characterization of Bph6-mediated resistance, the BPH insects showed significant preference between NIL-9311 and 9311 in 3 h and between NIL-NIP and Nipponbare in 120 h after release. BPH growth and development were inhibited, and the insect's survival rates were lower on Bph6-NIL plants, compared with the parents 9311 and Nipponbare. The results indicate that the Bph6 exerted prolonged antixenotic and antibiotic effects in Bph6-NIL plants, and NIL-9311 plants showed a quicker and stronger effect toward BPH than NIL-NIP plants.

Brown planthopper (Nilaparvata lugens Stål) is one of the major insect pests of rice. A Sri Lankan indica rice cultivar Rathu Heenati was found to be resistant to all biotypes of the brown planthopper. In the present study, a total of 268 F(7) RILs of IR50 and Rathu Heenati were phenotyped for their level of resistance against BPH by the standard seedbox screening test (SSST) in the greenhouse. A total of 53 SSR primers mapped on the chromosome 3 were used to screen the polymorphism between the parents IR50 and Rathu Heenati, out of which eleven were found to be polymorphic between IR50 and Rathu Heenati. The eleven primers that have shown polymorphism between the IR50 and Rathu Heenati parents were genotyped in a set of five resistant RILs and five susceptible RILs along with the parents for co-segregation analysis. Among the eleven primers, two primers namely RM3180 (18.22 Mb) and RM2453 (20.19 Mb) showed complete co-segregation with resistance. The identification of SSR markers linked with BPH resistant could be used for the maker assisted selection (MAS) program in rice breeding and to map the resistant genes on rice chromosomes for further gene cloning.

Rice yellow mottle virus (RYMV) is the most damaging rice-infecting virus in Africa. However, few sources of high resistance and only a single major resistance gene, RYMV1, are known to date. We screened a large representative collection of African cultivated rice (Oryza glaberrima) for RYMV resistance. Whereas high resistance is known to be very rare in Asian cultivated rice (Oryza sativa), we identified 29 (8%) highly resistant accessions in O. glaberrima. The MIF4G domain of RYMV1 was sequenced in these accessions. Some accessions possessed the rymv1-3 or rymv1-4 recessive resistance alleles previously described in O. glaberrima Tog5681 and Tog5672, respectively, and a new allele, rymv1-5, was identified, thereby increasing the number of resistance alleles in O. glaberrima to three. In contrast, only a single allele has been reported in O. sativa. Markers specific to the different alleles of the RYMV1 gene were developed for marker-assisted selection of resistant genotypes for disease management. In addition, the presence of the dominant susceptibility allele (Rymv1-1) in 15 resistant accessions suggests that their resistance is under different genetic control. An allelism test involving one of those accessions revealed a second major resistance gene, i.e., RYMV2. The diversity of resistance genes against RYMV in O. glaberrima species is discussed in relation to the diversification of the virus in Africa.

Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment. Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis, respectively. The binding activity of LTB protein expressed in transgenic rice callus to G(M1)-ganglioside, a receptor for biologically active LTB, was confirmed by G(M1)-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to G(M1)-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers. Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response. Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic E. coli heat-labile toxin.

Elicitors are molecules that stimulate defense responses in plants. Previously, an elicitor-encoding gene, named pemG1, was isolated from Magnaporthe grisea. To assess the function of pemG1 in rice (Oryza sativa L. cv. Nipponbare), the gene was cloned under a constitutive maize ubiquitin promoter and introduced into Nipponbare cultivar. The resultant plants showed stable integration and constitutive expression of the pemG1 gene. The expression of defense-related gene for phenylalanine ammonia-lyase was triggered and proline content was also increased in pemG1-expressing plants. The pemG1-expressing plants showed enhanced resistance against rice blast after inoculation with M. grisea spores, suggesting that the pemG1 expression enhances disease resistance in transgenic rice.

The Oryza sativa subsp. indica reference cultivar (cv.), 93-11 is completely resistant to many Chinese isolates of the rice blast fungus. Resistance segregated in a 3:1 (resistance/susceptible) ratio in an F(2) population from the cross between 93-11 and the japonica reference cv. Nipponbare, when challenged with two independent blast isolates. The chromosomal location of this monogenic resistance was mapped to a region of the long arm of chromosome 12 by bulk segregant analysis, using 180 evenly distributed SSR markers. Five additional SSR loci and nine newly developed PCR-based markers allowed the target region to be reduced to ca. 1.8 cM, equivalent in Nipponbare to about 800 kb. In the reference sequence of Nipponbare, this region includes an NBS-LRR cluster of four genes. The known blast resistance gene Pi-GD-3 also maps in this region, but the 93-11 resistance was distinguishable from Pi-GD-3 on the basis of race specificity. We have therefore named the 93-11 resistance Pi41. Seven markers completely linked to Pi41 will facilitate both marker-assisted breeding and gene isolation cloning.

Co-transformation of Oryza sativa L. var. Pusa Basmati1 was done using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector and a multi-copy binary vector in the same cell. The T-DNA of the cointegrate vector pGV2260::pSSJ1 carried the hygromycin phosphotransferase (hph) and beta-glucuronidase (gus) genes. The binary vector pCam-chi11, without a plant selectable marker gene, harboured the rice chitinase (chi11) gene under maize ubiquitin promoter. Co-transformation of the gene of interest (chi11) with the selectable marker gene (hph) occurred in 4 out of 20 T(0) plants (20%). Segregation of hph from chi11 was accomplished in two (CoT6 and CoT23) of the four co-transformed plants in the T(1) generation. The selectable marker-free (SMF) lines CoT6 and CoT23 harboured single copies of chi11. Homozygous SMF T(2) plants were established in the lines CoT6 and CoT23. Northern and Western blot analysis of the homozygous SMF lines showed high level of transgene expression. In comparison to untransformed controls, chitinase specific activity was 66- and 22-fold higher in the homozygous SMF T(2) plants of lines CoT6 and CoT23, respectively. The lines CoT6 and CoT23 exhibited 38 and 40% reduction in sheath blight disease, respectively.