Exploring genetic divergence and marker-trait associations for leaffolder Cnaphalocrocis medinalis (Guenee) resistance in rice landraces.

Rice production faces a significant threat from the rice leaffolder, Cnaphalocrocis medinalis. To address this challenge, growing resistant varieties stands out as a sustainable and eco-friendly pest management strategy. This necessitates identifying resistant sources and understanding their inheritance patterns through employing DNA markers for marker-assisted resistance breeding. Our study involves screening for resistant cultivars following the SES of IRRI, assessing genetic diversity among landraces using molecular markers, and identifying genomic regions associated with resistance. Screening indicated that 33.33%, 27.08%, 19.79%, and 19.80% of genotypes were resistant, moderately resistant, susceptible, and admixture, respectively. Landraces were categorized into three clusters, with clusters I and II predominantly containing moderately resistant and resistant cultivars, and cluster III mainly susceptible types. Molecular variance analysis revealed 12% variation among populations and 88% within the population. Simple linear regression identified significant marker-trait associations, with markers RM 162 and RM 284 on chromosomes 6 and 8, respectively, found highly associated with leaffolder resistance. Phenotypic variation in leaffolder damage correlated highly with the allelic effects of these markers. Further confirmation of marker linkage with resistance loci was established through independent assays on highly resistant and susceptible genotypes. The information derived from genetic diversity and marker-trait associations will be useful for future marker-assisted resistance breeding programs, enhancing the sustainability of rice production.

iTRAQ based proteomic analysis of rice lines having single or stacked blast resistance genes: Pi54/Pi54rh during incompatible interaction with Magnaporthe oryzae.

Deployment of single or multiple blast resistance (R) genes in rice plant is considered to be the most promising approach to enhance resistance against blast disease caused by fungus Magnaporthe oryzae. At the proteome level, relatively little information about R gene mediated defence mechanisms for single and stacking resistance characteristics is available. The overall objective of this study is to look at the proteomics of rice plants that have R genes; Pi54, Pi54rh and stacked Pi54 + Pi54rh in response to rice blast infection. In this study 'isobaric tag for relative and absolute quantification' (iTRAQ)-based proteomics analysis was performed in rice plants at 72-h post inoculation with Magnaporthe oryzae and various differentially expressed proteins were identified in these three transgenic lines in comparison to wild type during resistance response to blast pathogen. Through STRING analysis, the observed proteins were further examined to anticipate their linked partners, and it was shown that several defense-related proteins were co-expressed. These proteins can be employed as targets in future rice resistance breeding against Magnaporthe oryzae. The current study is the first to report a proteomics investigation of rice lines that express single blast R gene Pi54, Pi54rh and stacked (Pi54 + Pi54rh) during incompatible interaction with Magnaporthe oryzae. The differentially expressed proteins indicated that secondary metabolites, reactive oxygen species-related proteins, phenylpropanoid, phytohormones and pathogenesis-related proteins have a substantial relationship with the defense response against Magnaporthe oryzae. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01327-3.

Exploring the diversity of virulence genes in the Magnaporthe population infecting millets and rice in India.

Blast pathogen, Magnaporthe spp., that infects ancient millet crops such pearl millet, finger millet, foxtail millet, barnyard millet, and rice was isolated from different locations of blast hotspots in India using single spore isolation technique and 136 pure isolates were established. Numerous growth characteristics were captured via morphogenesis analysis. Among the 10 investigated virulent genes, we could amplify MPS1 (TTK Protein Kinase) and Mlc (Myosin Regulatory Light Chain edc4) in majority of tested isolates, regardless of the crop and region where they were collected, indicating that these may be crucial for their virulence. Additionally, among the four avirulence (Avr) genes studied, Avr-Pizt had the highest frequency of occurrence, followed by Avr-Pia. It is noteworthy to mention that Avr-Pik was present in the least number of isolates (9) and was completely absent from the blast isolates from finger millet, foxtail millet, and barnyard millet. A comparison at the molecular level between virulent and avirulent isolates indicated observably large variation both across (44%) and within (56%) them. The 136 Magnaporthe spp isolates were divided into four groups using molecular markers. Regardless of their geographic distribution, host plants, or tissues affected, the data indicate that the prevalence of numerous pathotypes and virulence factors at the field level, which may lead to a high degree of pathogenic variation. This research could be used for the strategic deployment of resistant genes to develop blast disease-resistant cultivars in rice, pearl millet, finger millet, foxtail millet, and barnyard millet.

Understanding complex genetic architecture of rice grain weight through QTL-meta analysis and candidate gene identification.

Quantitative trait loci (QTL) for rice grain weight identified using bi-parental populations in various environments were found inconsistent and have a modest role in marker assisted breeding and map-based cloning programs. Thus, the identification of a consistent consensus QTL region across populations is critical to deploy in marker aided breeding programs. Using the QTL meta-analysis technique, we collated rice grain weight QTL information from numerous studies done across populations and in diverse environments to find constitutive QTL for grain weight. Using information from 114 original QTL in meta-analysis, we discovered three significant Meta-QTL (MQTL) for grain weight on chromosome 3. According to gene ontology, these three MQTL have 179 genes, 25 of which have roles in developmental functions. Amino acid sequence BLAST of these genes indicated their orthologue conservation among core cereals with similar functions. MQTL3.1 includes the OsAPX1, PDIL, SAUR, and OsASN1 genes, which are involved in grain development and have been discovered to play a key role in asparagine biosynthesis and metabolism, which is crucial for source-sink regulation. Five potential candidate genes were identified and their expression analysis indicated a significant role in early grain development. The gene sequence information retrieved from the 3 K rice genome project revealed the deletion of six bases coding for serine and alanine in the last exon of OsASN1 led to an interruption in the synthesis of α-helix of the protein, which negatively affected the asparagine biosynthesis pathway in the low grain weight genotypes. Further, the MQTL3.1 was validated using linked marker RM7197 on a set of genotypes with extreme phenotypes. MQTL that have been identified and validated in our study have significant scope in MAS breeding and map-based cloning programs for improving rice grain weight.

Versatile role of silicon in cereals: Health benefits, uptake mechanism, and evolution.

Silicon (Si) is an omnipresent and second most abundant element in the soil lithosphere after oxygen. Silicon being a beneficial element imparts several benefits to the plants and animals. In many plant species, including the cereals the uptake of Si from the soil even exceeds the uptake of essential nutrients. Cereals are the monocots which are known to accumulate a high amount of Si, and reaping maximum benefits associated with it. Cereals contribute a high amount of Si to the human diet compared to other food crops. In the present review, we have summarized distribution of the dietary Si in cereals and its role in the animal and human health. The Si derived benefits in cereals, specifically with respect to biotic and abiotic stress tolerance has been described. We have also discussed the molecular mechanism involved in the Si uptake in cereals, evolution of the Si transport mechanism and genetic variation in the Si concentration among different cultivars of the same species. Various genetic mutants deficient in the Si uptake have been developed and many QTLs governing the Si accumulation have been identified in cereals. The existing knowledge about the Si biology and available resources needs to be explored to understand and improve the Si accumulation in crop plants to achieve sustainability in agriculture.

Deciphering the role of microRNAs during Pi54 gene mediated Magnaporthe oryzae resistance response in rice.

The broad-spectrum resistance gene Pi54 confers resistance to multiple isolates of Magnaporthe oryzae in rice. In order to decipher the molecular mechanism underlying the Pi54 mediated resistance in rice line Taipei309 Pi54 (carrying Pi54), miRNAome study was performed at 24 h post-inoculation (hpi) with M. oryzae. A total of 222 known miRNAs representing 101 miRNA families were found in this study. Of these, 29 and 24 miRNAs were respectively up- and down-regulated in the resistant Taipei309 Pi54 . Defence response (DR) genes, like, NBSGO35, and OsWAK129b, and genes related to transcription factors were up-regulated in Taipei309 Pi54 line. The vast array of miRNA candidates identified here are miR159c, miR167c, miR2100, miR2118o, miR2118l, miR319a, miR393, miR395l, miR397a, miR397b, miR398, miR439g, miR531b, miR812f, and miR815c, and they manifest their role in balancing the interplay between various DR genes during Pi54 mediated resistance. We also validated miRNA/target gene pairs involved in hormone signalling, and cross-talk among hormone pathways regulating the rice immunity. This study suggests that the Pi54 gene mediated blast resistance is influenced by several microRNAs through PTI and ETI components in the rice line Taipei309 Pi54 , leading to incompatible host-pathogen interaction.

Fascinating role of silicon to combat salinity stress in plants: An updated overview.

Salt stress limits plant growth and productivity by severely impacting the fundamental physiological processes. Silicon (Si) supplementation is considered one of the promising methods to improve plant resilience under salt stress. Here, the role of Si in modulating physiological and biochemical processes that get adversely affected by high salinity, is discussed. Although numerous reports show the beneficial effects of Si under stress, the precise molecular mechanism underlying this is not well understood. Questions like whether all plants are equally benefitted with Si supplementation despite having varying Si uptake capability and salinity tolerance are still elusive. This review illustrates the Si uptake and accumulation mechanism to understand the direct or indirect participation of Si in different physiological processes. Evaluation of plant responses at transcriptomics and proteomics levels are promising in understanding the role of Si. Integration of physiological understanding with omics scale information highlighted Si supplementation affecting the phytohormonal and antioxidant responses under salinity as a key factor defining improved resilience. Similarly, the crosstalk of Si with lignin and phenolic content under salt stress also seems to be an important phenomenon helping plants to reduce the stress. The present review also addressed various crucial mechanisms by which Si application alleviates salt stress, such as a decrease in oxidative damage, decreased lipid peroxidation, improved photosynthetic ability, and ion homeostasis. Besides, the application and challenges of using Si-nanoparticles have also been addressed. Comprehensive information and discussion provided here will be helpful to better understand the role of Si under salt stress.

Blast resistance gene Pi54 over-expressed in rice to understand its cellular and sub-cellular localization and response to different pathogens.

Rice blast resistance gene, Pi54 provides broad-spectrum resistance against different strains of Magnaporthe oryzae. Understanding the cellular localization of Pi54 protein is an essential step towards deciphering its place of interaction with the cognate Avr-gene. In this study, we investigated the sub-cellular localization of Pi54 with Green Fluorescent Protein (GFP) as a molecular tag through transient and stable expression in onion epidermal cells (Allium cepa) and susceptible japonica cultivar rice Taipei 309 (TP309), respectively. Confocal microscopy based observations of the onion epidermal cells revealed nucleus and cytoplasm specific GFP signals. In the stable transformed rice plants, GFP signal was recorded in the stomata, upper epidermal cells, mesophyll cells, vascular bundle, and walls of bundle sheath and bulliform cells of leaf tissues. These observations were further confirmed by Immunocytochemical studies. Using GFP specific antibodies, it was found that there was sufficient aggregation of GFP::Pi54protein in the cytoplasm of the leaf mesophyll cells and periphery of the epidermal cells. Interestingly, the transgenic lines developed in this study could show a moderate level of resistance to Xanthomonas oryzae and Rhizoctonia solani, the causal agents of the rice bacterial blight and sheath blight diseases, respectively. This study is a first detailed report, which emphasizes the cellular and subcellular distribution of the broad spectrum blast resistance gene Pi54 in rice and the impact of its constitutive expression towards resistance against other fungal and bacterial pathogens of rice.

Corrigendum: Cloning and functional validation of early inducible Magnaporthe oryzae responsive CYP76M7 promoter from rice.

[This corrects the article on p. 371 in vol. 6, PMID: 26052337.].

Stacking of blast resistance orthologue genes in susceptible indica rice line improves resistance against Magnaporthe oryzae.

The emergence of new strains of Magnaporthe oryzae (M. oryzae) is associated with recurrent failure of resistance response mediated by single resistance (R) gene in rice. Therefore, stacking or combining of multiple R genes could improve the durability of resistance against multiple strains of M. oryzae. To achieve this, in the present study, intragenic stacking of rice blast resistance orthologue genes Pi54 and Pi54rh was performed through co-transformation approach. Both these genes were expressed under the control of independent promoters and blast susceptible indica rice line IET17021 was used for transformation. The highly virulent M. oryzae strain Mo-ei-ger1 that could knock down most of the major single blast R genes including Pi54 and exhibiting 89% virulence spectrum was used for phenotypic analysis. The stacked transgenic IET17021 lines (Pi54 + Pi54rh) have shown complete resistance to Mo-ei-ger1 strain in comparison to non-transgenic lines. These two R gene stacked indica transgenic lines could serves as a novel germplasm for rice blast resistance breeding programmes.

Status and Prospects of Next Generation Sequencing Technologies in Crop Plants.

The history of DNA sequencing dates back to 1970s. During this period the two first generation nucleotide sequencing techniques were developed. Subsequently the Sanger's dideoxy method of sequencing gained popularity over Maxam and Gilbert's chemical method of sequencing. However, in the last decade, we have observed revolutionary changes in DNA sequencing technologies leading to the emergence of next-generation sequencing (NGS) techniques. NGS technologies have enhanced the throughput and speed of sequencing combined with bringing down the overall cost of the process over a time. The major applications of NGS technologies being genome sequencing and resequencing, transcriptomics, metagenomics in relation to plant-microbe interactions, exon and genome capturing, development of molecular markers and evolutionary studies. In this review, we present a broader picture of evolution of NGS tools, its various applications in crop plants, and future prospects of the technology for crop improvement.

Co-transformation mediated stacking of blast resistance genes Pi54 and Pi54rh in rice provides broad spectrum resistance against Magnaporthe oryzae.

KEY MESSAGE: This is the first report of stacking two major blast resistance genes in blast susceptible rice variety using co-transformation method to widen the resistance spectrum against different isolates of Magnaporthe oryzae. Single resistance (R-) gene mediated approach for the management of rice blast disease has met with frequent breakdown in resistance response. Besides providing the durable resistance, gene pyramiding or stacking also imparts broad spectrum resistance against plant pathogens, including rice blast. In the present study, we stacked two R-genes; Pi54 and Pi54rh having broad spectrum resistance against multiple isolates of Magnaporthe oryzae (M. oryzae). Both Pi54 and Pi54rh expressed under independent promoters were transferred into the blast susceptible japonica rice Taipei 309 (TP309) using particle gun bombardment method. Functional complementation analysis of stacked transgenic rice lines showed higher level of resistance to a set of highly virulent M. oryzae isolates collected from different rice growing regions. qRT-PCR analysis has shown M. oryzae induced expression of both the R-genes in stacked transgenic lines. The present study also demonstrated the effectiveness of the strategy for rapid single step gene stacking using co-transformation approach to engineer durable resistance against rice blast disease and also this is the first report in which two blast R-genes are stacked together using co-transformation approach. The two-gene-stacked transgenic line developed in this study can be used further to understand the molecular aspects of defense-related pathways vis-a-vis single R-gene containing transgenic lines.

Host Delivered RNAi, an efficient approach to increase rice resistance to sheath blight pathogen (Rhizoctonia solani).

Rhizoctonia solani, the causal agent of rice sheath blight disease, causes significant losses worldwide as there are no cultivars providing absolute resistance to this fungal pathogen. We have used Host Delivered RNA Interference (HD-RNAi) technology to target two PATHOGENICITY MAP KINASE 1 (PMK1) homologues, RPMK1-1 and RPMK1-2, from R. solani using a hybrid RNAi construct. PMK1 homologues in other fungal pathogens are essential for the formation of appressorium, the fungal infection structures required for penetration of the plant cuticle, as well as invasive growth once inside the plant tissues and overall viability of the pathogen within the plant. Evaluation of transgenic rice lines revealed a significant decrease in fungal infection levels compared to non-transformed controls and the observed delay in disease symptoms was further confirmed through microscopic studies. Relative expression levels of the targeted genes, RPMK1-1 and RPMK1-2, were determined in R. solani infecting either transgenic or control lines with significantly lower levels observed in R. solani infecting transgenic lines carrying the HD-RNAi constructs. This is the first report demonstrating the effectiveness of HD-RNAi against sheath blight and offers new opportunities for durable control of the disease as it does not rely on resistance conferred by major resistance genes.

Novel Chitinase Gene LOC_Os11g47510 from Indica Rice Tetep Provides Enhanced Resistance against Sheath Blight Pathogen Rhizoctonia solani in Rice.

Sheath blight disease (ShB), caused by the fungus Rhizoctonia solani Kühn, is one of the most destructive diseases of rice (Oryza sativa L.), causing substantial yield loss in rice. In the present study, a novel rice chitinase gene, LOC_Os11g47510 was cloned from QTL region of R. solani tolerant rice line Tetep and used for functional validation by genetic transformation of ShB susceptible japonica rice line Taipei 309 (TP309). The transformants were characterized using molecular and functional approaches. Molecular analysis by PCR using a set of primers specific to CaMv 35S promoter, chitinase and HptII genes confirmed the presence of transgene in transgenic plants which was further validated by Southern hybridization. Further, qRT-PCR analysis of transgenic plants showed good correlation between transgene expression and the level of sheath blight resistance among transformants. Functional complementation assays confirmed the effectiveness of the chitinase mediated resistance in all the transgenic TP309 plants with varying levels of enhanced resistance against R. solani. Therefore, the novel chitinase gene cloned and characterized in the present study from the QTL region of rice will be of significant use in molecular plant breeding program for developing sheath blight resistance in rice.

Dissection of genomic features and variations of three pathotypes of Puccinia striiformis through whole genome sequencing.

Stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici, is one of the important diseases of wheat. We used NGS technologies to generate a draft genome sequence of two highly virulent (46S 119 and 31) and a least virulent (K) pathotypes of P. striiformis from the Indian subcontinent. We generated ~24,000-32,000 sequence contigs (N50;7.4-9.2 kb), which accounted for ~86X-105X sequence depth coverage with an estimated genome size of these pathotypes ranging from 66.2-70.2 Mb. A genome-wide analysis revealed that pathotype 46S 119 might be highly evolved among the three pathotypes in terms of year of detection and prevalence. SNP analysis revealed that ~47% of the gene sets are affected by nonsynonymous mutations. The extracellular secreted (ES) proteins presumably are well conserved among the three pathotypes, and perhaps purifying selection has an important role in differentiating pathotype 46S 119 from pathotypes K and 31. In the present study, we decoded the genomes of three pathotypes, with 81% of the total annotated genes being successfully assigned functional roles. Besides the identification of secretory genes, genes essential for pathogen-host interactions shall prove this study as a huge genomic resource for the management of this disease using host resistance.

Draft Genome of the Wheat Rust Pathogen (Puccinia triticina) Unravels Genome-Wide Structural Variations during Evolution.

Leaf rust is one of the most important diseases of wheat and is caused by Puccinia triticina, a highly variable rust pathogen prevalent worldwide. Decoding the genome of this pathogen will help in unraveling the molecular basis of its evolution and in the identification of genes responsible for its various biological functions. We generated high quality draft genome sequences (approximately 100- 106 Mb) of two races of P. triticina; the variable and virulent Race77 and the old, avirulent Race106. The genomes of races 77 and 106 had 33X and 27X coverage, respectively. We predicted 27678 and 26384 genes, with average lengths of 1,129 and 1,086 bases in races 77 and 106, respectively and found that the genomes consisted of 37.49% and 39.99% repetitive sequences. Genome wide comparative analysis revealed that Race77 differs substantially from Race106 with regard to segmental duplication (SD), repeat element, and SNP/InDel characteristics. Comparative analyses showed that Race 77 is a recent, highly variable and adapted Race compared with Race106. Further sequence analyses of 13 additional pathotypes of Race77 clearly differentiated the recent, active and virulent, from the older pathotypes. Average densities of 2.4 SNPs and 0.32 InDels per kb were obtained for all P. triticina pathotypes. Secretome analysis demonstrated that Race77 has more virulence factors than Race 106, which may be responsible for the greater degree of adaptation of this pathogen. We also found that genes under greater selection pressure were conserved in the genomes of both races, and may affect functions crucial for the higher levels of virulence factors in Race77. This study provides insights into the genome structure, genome organization, molecular basis of variation, and pathogenicity of P. triticina The genome sequence data generated in this study have been submitted to public domain databases and will be an important resource for comparative genomics studies of the more than 4000 existing Puccinia species.

Functional Characterization of Novel Chitinase Genes Present in the Sheath Blight Resistance QTL: qSBR11-1 in Rice Line Tetep.

Rice sheath blight disease caused by Rhizoctonia solani is one of the most devastating diseases in rice leading to heavy yield losses. Due to the polygenic nature of resistance, no major resistance gene with complete host resistance against R. solani has been reported. In this study, we have performed molecular and functional analysis of the genes associated with the major R. solani-resistance QTL qSBR11-1 in the indica rice line Tetep. Sequence analysis revealed the presence of a set of 11 tandem repeats containing genes with a high degree of homology to class III chitinase defense response genes. Real-time quantitative PCR analysis showed that all the genes are strongly induced 36 h after R. solani infection. Comparison between the resistant Tetep and the susceptible HP2216 lines shows that the induction of the chitinase genes is much higher in the Tetep line. Recombinant protein produced in vitro for six of the eleven genes showed chitinolytic activity in gel assays but we did not detect any xylanase inhibitory activity. All the six in vitro expressed proteins show antifungal activity with a clear inhibitory effect on the growth of the R. solani mycelium. The characterized chitinase genes can provide an important resource for the genetic improvement of R. solani susceptible rice lines for sheath blight resistance breeding.

Oscillating Transcriptome during Rice-Magnaporthe Interaction.

Rice blast disease caused by the fungus, Magnaporthe oryzae, is one of the most devastating diseases of rice. Deciphering molecular mechanism of host-pathogen interactions is of great importance in devising disease management strategies. Transcription being the first step for gene regulation in eukaryotes, basic understanding of the transcriptome is sine qua non for devising effective management strategy. The availability of genome sequences of rice and M. oryzae has facilitated the process to a large extent. The current review summarizes recent understanding of rice-blast pathosystem, application of transcriptomics approaches to understand the interactions employing different platforms, major determinants in the interaction and possibility of using certain candidate for conditioning enhanced disease resistance (Effector Triggered Immunity and PAMP Triggered Immunity) and downstream signalling in rice. A better understanding of the interaction elements and effective strategies hold potential to reduce yield losses in rice caused by M. oryzae.

A web-based microsatellite database for the Magnaporthe oryzae genome.

Microsatellites have been widely utilized for molecular marker development. Codominant and multiallelic nature of these simple repeats have several advantages over other types of molecular markers. Their broad applicability in the area of molecular biology like gene mapping, genome characterization, genome evolution, and gene regulation has been reported in various crop plants, animals and fungi. Considering these benefits of the SSR markers, a MMDB (Magnaporthe oryzae Microsatellite Database) was developed to help in understanding about the pathogen and its diversity at strains level of a particular geographic region, which can help us to make a proper utilization of blast resistance genes in the region. This microsatellite database is based on whole genome sequence of two M. oryzae isolates, RML-29 (2665 SSRs from 43037792 bp) and RP-2421 (3169 SSRs from 45510614 bp). Although, first M. oryzae genome (70-15) was sequenced in 2005, but this sequenced isolate is not a true field isolate of M. oryzae. Therefore, MMDB has great potential in the study of diversification and characterization of M. oryzae and other related fungi. AVAILABILITY: http://14.139.229.199/home.aspx.

Cloning and functional validation of early inducible Magnaporthe oryzae responsive CYP76M7 promoter from rice.

Cloning and functional characterization of plant pathogen inducible promoters is of great significance for their use in the effective management of plant diseases. The rice gene CYP76M7 was up regulated at 24, 48, and 72 hours post inoculation (hpi) with two isolates of Magnaporthe oryzae Mo-ei-11 and Mo-ni-25. In this study, the promoter of CYP76M7 gene was cloned from rice cultivar HR-12, characterized and functionally validated. The Transcription Start Site of CYP76M7 was mapped at 45 bases upstream of the initiation codon. To functionally validate the promoter, 5' deletion analysis of the promoter sequences was performed and the deletion fragments fused with the ß-glucuronidase (GUS) reporter gene were used for generating stable transgenic Arabidopsis plants as well as for transient expression in rice. The spatial and temporal expression pattern of GUS in transgenic Arabidopsis plants and also in transiently expressed rice leaves revealed that the promoter of CYP76M7 gene was induced by M. oryzae. The induction of CYP76M7 promoter was observed at 24 hpi with M. oryzae. We report that, sequences spanning -222 bp to -520 bp, with the cluster of three W-boxes, two ASF1 motifs and a single GT-1 element may contribute to the M. oryzae inducible nature of CYP76M7 promoter. The promoter characterized in this study would be an ideal candidate for the overexpression of defense genes in rice for developing durable blast resistance rice lines.