Oschib1 gene encoding a GH18 chitinase confers resistance against sheath blight disease of rice caused by Rhizoctonia solani AG1-IA.

Sheath blight disease of rice caused by Rhizoctonia solani AG1-IA, is a major fungal disease responsible for huge loss to grain yield and quality. The major limitation of achieving persistent and reliable resistance against R. solani is the governance of disease resistance trait by many genes. Therefore, functional characterization of new genes involved in sheath blight resistance is necessary to understand the mechanism of resistance as well as evolving effective strategies to manage the disease through host-plant resistance. In this study, we performed RNA sequencing of six diverse rice genotypes (TN1, BPT5204, Vandana, N22, Tetep, and Pankaj) from sheath and leaf tissue of control and fungal infected samples. The approach for identification of candidate resistant genes led to identification of 352 differentially expressed genes commonly present in all the six genotypes. 23 genes were analyzed for RT-qPCR expression which helped identification of Oschib1 showing differences in expression level in a time-course manner between susceptible and resistant genotypes. The Oschib1 encoding classIII chitinase was cloned from resistant variety Tetep and over-expressed in susceptible variety Taipei 309. The over-expression lines showed resistance against R. solani, as analyzed by detached leaf and whole plant assays. Interestingly, the resistance response was correlated with the level of transgene expression suggesting that the enzyme functions in a dose dependent manner. We report here the classIIIb chitinase from chromosome10 of rice showing anti-R. solani activity to combat the dreaded sheath blight disease.

Fine mapping and sequence analysis reveal a promising candidate gene encoding a novel NB-ARC domain derived from wild rice (Oryza officinalis) that confers bacterial blight resistance.

Bacterial blight disease of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious constraints in rice production. The most sustainable strategy to combat the disease is the deployment of host plant resistance. Earlier, we identified an introgression line, IR 75084-15-3-B-B, derived from Oryza officinalis possessing broad-spectrum resistance against Xoo. In order to understand the inheritance of resistance in the O. officinalis accession and identify genomic region(s) associated with resistance, a recombinant inbred line (RIL) mapping population was developed from the cross Samba Mahsuri (susceptible to bacterial blight) × IR 75084-15-3-B-B (resistant to bacterial blight). The F2 population derived from the cross segregated in a phenotypic ratio of 3: 1 (resistant susceptible) implying that resistance in IR 75084-15-3-B-B is controlled by a single dominant gene/quantitative trait locus (QTL). In the F7 generation, a set of 47 homozygous resistant lines and 47 homozygous susceptible lines was used to study the association between phenotypic data obtained through screening with Xoo and genotypic data obtained through analysis of 7K rice single-nucleotide polymorphism (SNP) chip. Through composite interval mapping, a major locus was detected in the midst of two flanking SNP markers, viz., Chr11.27817978 and Chr11.27994133, on chromosome 11L with a logarithm of the odds (LOD) score of 10.21 and 35.93% of phenotypic variation, and the locus has been named Xa48t. In silico search in the genomic region between the two markers flanking Xa48t identified 10 putatively expressed genes located in the region of interest. The quantitative expression and DNA sequence analysis of these genes from contrasting parents identified the Os11g0687900 encoding an NB-ARC domain-containing protein as the most promising gene associated with resistance. Interestingly, a 16-bp insertion was noticed in the untranslated region (UTR) of the gene in the resistant parent, IR 75084-15-3-B-B, which was absent in Samba Mahsuri. The association of Os11g0687900 with resistance phenotype was further established by sequence-based DNA marker analysis in the RIL population. A co-segregating PCR-based INDEL marker, Marker_Xa48, has been developed for use in the marker-assisted breeding of Xa48t.

Spatio-temporal expression pattern of Raffinose Synthase genes determine the levels of Raffinose Family Oligosaccharides in peanut (Arachis hypogaea L.) seed.

Raffinose family oligosaccharides (RFOs) are known to have important physiological functions in plants. However, the presence of RFOs in legumes causes flatulence, hence are considered antinutrients. To reduce the RFOs content to a desirable limit without compromising normal plant development and functioning, the identification of important regulatory genes associated with the biosynthetic pathway is a prerequisite. In the present study, through comparative RNA sequencing in contrasting genotypes for seed RFOs content at different seed maturity stages, differentially expressed genes (DEGs) associated with the pathway were identified. The DEGs exhibited spatio-temporal expression patterns with high RFOs variety showing early induction of RFOs biosynthetic genes and low RFOs variety showing a late expression at seed maturity. Selective and seed-specific differential expression of raffinose synthase genes (AhRS14 and AhRS6) suggested their regulatory role in RFOs accumulation in peanut seeds, thereby serving as promising targets in low RFOs peanut breeding programs. Despite stachyose being the major seed RFOs fraction, differential expression of raffinose synthase genes indicated the complex metabolic regulation of this pathway. The transcriptomic resource and the genes identified in this study could be studied further to develop low RFOs varieties, thus improving the overall nutritional quality of peanuts.

Whole-Genome Sequencing of KMR3 and Oryza rufipogon-Derived Introgression Line IL50-13 (Chinsurah Nona 2/Gosaba 6) Identifies Candidate Genes for High Yield and Salinity Tolerance in Rice.

The genomes of an elite rice restorer line KMR3 (salinity-sensitive) and its salinity-tolerant introgression line IL50-13, a popular variety of coastal West Bengal, India, were sequenced. High-quality paired-end reads were obtained for KMR3 (147.6 million) and IL50-13 (131.4 million) with a sequencing coverage of 30X-39X. Scaffolds generated from the pre-assembled contigs of each sequenced genome were mapped separately onto the reference genome of Oryza sativa ssp. japonica cultivar Nipponbare to identify genomic variants in terms of SNPs and InDels. The SNPs and InDels identified for KMR3 and IL50-13 were then compared with each other to identify polymorphic SNPs and InDels unique and common to both the genomes. Functional enrichment analysis of the protein-coding genes with unique InDels identified GO terms involved in protein modification, ubiquitination, deubiquitination, peroxidase activity, and antioxidant activity in IL50-13. Linoleic acid metabolism, circadian rhythm, and alpha-linolenic acid metabolism pathways were enriched in IL50-13. These GO terms and pathways are involved in reducing oxidative damage, thus suggesting their role in stress responses. Sequence analysis of QTL markers or genes known to be associated with grain yield and salinity tolerance showed polymorphism in 20 genes, out of which nine were not previously reported. These candidate genes encoded Nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC) domain-containing protein, cyclase, receptor-like kinase, topoisomerase II-associated protein PAT1 domain-containing protein, ion channel regulatory protein, UNC-93 domain-containing protein, subunit A of the heteromeric ATP-citrate lyase, and three conserved hypothetical genes. Polymorphism was observed in the coding, intron, and untranslated regions of the genes on chromosomes 1, 2, 4, 7, 11, and 12. Genes showing polymorphism between the two genomes were considered as sequence-based new candidates derived from Oryza rufipogon for conferring high yield and salinity tolerance in IL50-13 for further functional studies.

Revealing the effect of seed phosphorus concentration on seedling vigour and growth of rice using mutagenesis approach.

The harvested plant products, specifically, the grains of cereals are major drivers of soil phosphorus (P) depletion. However, the breeding or biotechnology efforts to develop low P seeds have not been attempted because of possible adverse effects on seedling vigour and crop establishment. Several studies have contradictory observations on influence of seed P on seedling vigour. Lack of appropriate genetic material has been the major bottleneck in reaching the consensus. In this study, we used 30 EMS induced mutants of rice cultivar Nagina22 to understand the role of seed P on seedling vigour and associated physiological processes. Seedling vigour, morpho-physiological characteristics, acid phosphatases, alpha-amylase, and expression of P transporter genes were analyzed in seedlings obtained from seeds of high and low grain P mutants. The study suggests that seed P has a significant role on seedling vigour, chlorophyll content and photosynthesis process of young seedlings, and P transport from roots. Notably, we identified few mutants such as NH4791, NH4785, NH4714, NH4663, NH4614, and NH4618 which showed least influence of low seed P on seedling vigour and other metabolic processes. Therefore, these mutants can be used in breeding programs aiming for development of low P grains. Also, these and other identified mutants can be used to decipher the genetic and molecular mechanisms regulating the differential response of seed P on germination, seedling vigour and several other physiological processes influencing the crop growth and establishment.

Genetic, Epigenetic, Genomic and Microbial Approaches to Enhance Salt Tolerance of Plants: A Comprehensive Review.

Globally, soil salinity has been on the rise owing to various factors that are both human and environmental. The abiotic stress caused by soil salinity has become one of the most damaging abiotic stresses faced by crop plants, resulting in significant yield losses. Salt stress induces physiological and morphological modifications in plants as a result of significant changes in gene expression patterns and signal transduction cascades. In this comprehensive review, with a major focus on recent advances in the field of plant molecular biology, we discuss several approaches to enhance salinity tolerance in plants comprising various classical and advanced genetic and genetic engineering approaches, genomics and genome editing technologies, and plant growth-promoting rhizobacteria (PGPR)-based approaches. Furthermore, based on recent advances in the field of epigenetics, we propose novel approaches to create and exploit heritable genome-wide epigenetic variation in crop plants to enhance salinity tolerance. Specifically, we describe the concepts and the underlying principles of epigenetic recombinant inbred lines (epiRILs) and other epigenetic variants and methods to generate them. The proposed epigenetic approaches also have the potential to create additional genetic variation by modulating meiotic crossover frequency.

Genome-Wide Expression Profiling of Small RNAs in Indian Strain of Rhizoctonia solani AG1-1A Reveals Differential Regulation of milRNAs during Pathogenesis and Crosstalk of Gene Regulation.

Rhizoctonia solani AG1-1A is a necrotrophic fungus that causes sheath blight disease in rice. The reliable resistant source against this phytopathogenic fungus is not available in the gene pool of rice. Better understanding of pathogen genomics and gene regulatory networks are critical to devise alternate strategies for developing resistance against this noxious pathogen. In this study, miRNA-like RNAs (milRNAs) of an Indian strain of R. solani were identified by deep sequencing of small RNAs. We identified 128 known and 22 novel milRNAs from 20,963,123 sequence reads. These milRNAs showed 1725 target genes in the fungal genome which include genes associated with growth, development, pathogenesis and virulence of R. solani. Notably, these fungal milRNAs showed their target genes in host (rice) genome also which were later verified by qRT-PCR. The host target genes are associated with auxin metabolism, hypersensitive response, defense genes, and genes related to growth and development of rice. Osa-vacuolar-sorting receptor precursor: Rhi-milR-13, Osa-KANADI1:Rhi-milR-124, Osa-isoflavone reductase: Rhi-milR-135, Osa-nuclear transcription factor Y:Rhi-milR-131, Osa-NB-ARC domain containing protein: Rhi-milR-18, and Osa-OsFBX438: Rhi-milR-142 are notable potential regulons of host target genes: fungal milRNAs that need to be investigated for better understanding of the crosstalk of RNAi pathways between R. solani and rice. The detailed expression analysis of 17 milRNAs by qRT-PCR was analysed during infection at different time points of inoculation, at different growth stages of the host, in four different genotypes of the host, and also in four different strains of fungi which revealed differential regulation of milRNAs associated with pathogenesis and virulence. This study highlights several important findings on fungal milRNAs which need to be further studied and characterized to decipher the gene expression and regulation of this economically important phytopathogen.

PAP90, a novel rice protein plays a critical role in regulation of D1 protein stability of PSII.

Introduction: Photosystem II (PSII) protein complex plays an essential role in the entire photosynthesis process. Various known and unknown protein factors are involved in the dynamics of the PSII complex that need to be characterized in crop plants for enhancing photosynthesis efficiency and productivity. Objectives: The experiments were conducted to decipher the regulatory proteins involved in PSII dynamics of rice crop. Methods: A novel rice regulatory protein PAP90 (PSII auxiliary protein ~90 kDa) was characterized by generating a loss-of-function mutant pap90. The mutation was characterized at molecular level followed by various experiments to analyze the morphological, physiological and biochemical processes of mutant under control and abiotic stresses. Results: The pap90 mutant showed reduced photosynthesis due to D1 protein instability that subsequently causes inadequate accumulation of thylakoid membrane complexes, especially PSII and decreases PSII functional efficiency. Expression of OsFtsH family genes and proteins were induced in the mutant, which are known to play a key role in D1 protein degradation and turnover. The reduced D1 protein accumulation in the mutant increased the production of reactive oxygen species (ROS). The accumulation of ROS along with the increased activity of antioxidant enzymes and induced expression of stress-associated genes and proteins in pap90 mutant contributed to its water-limited stress tolerance ability. Conclusion: We propose that PAP90 is a key auxiliary protein that interacts with D1 protein and maintains its stability, thereby promoting subsequent assembly of the PSII and associated membrane complexes.

Regulatory non-coding RNAs: a new frontier in regulation of plant biology.

Beyond the most crucial roles of RNA molecules as a messenger, ribosomal, and transfer RNAs, the regulatory role of many non-coding RNAs (ncRNAs) in plant biology has been recognized. ncRNAs act as riboregulators by recognizing specific nucleic acid targets through homologous sequence interactions to regulate plant growth, development, and stress responses. Regulatory ncRNAs, ranging from small to long ncRNAs (lncRNAs), exert their control over a vast array of biological processes. Based on the mode of biogenesis and their function, ncRNAs evolved into different forms that include microRNAs (miRNAs), small interfering RNAs (siRNAs), miRNA variants (isomiRs), lncRNAs, circular RNAs (circRNAs), and derived ncRNAs. This article explains the different classes of ncRNAs and their role in plant development and stress responses. Furthermore, the applications of regulatory ncRNAs in crop improvement, targeting agriculturally important traits, have been discussed.

NH787 EMS mutant of rice variety Nagina22 exhibits higher phosphate use efficiency.

Rice (Oryza sativa L.), a major dietary source, is often cultivated in soils poor in available inorganic orthophosphate (Pi), which is a key nutrient for growth and development. Poor soils are amended by phosphorus (P) fertilizer, which is derived from the non-renewable rock phosphate reserves. Therefore, there is a need for developing rice varieties with high productivity under low P conditions. At the ICAR-IIRR, ethyl methanesulfonate (EMS) mutagenized rice genotype Nagina22 (N22) were screened for high grain yield in Pi-deprived soil, which led to the identification of ~ 10 gain-of-function mutants including NH787. Here, detailed comparative morphophysiological, biochemical, and molecular analyses of N22 and NH787 were carried out in hydroponics and potting soil under different Pi regimes. Under Pi-deprived condition, compared with N22, NH787 exhibited higher root and vegetative biomass, the number of tillers, and grain yield. The augmented agronomic traits of NH787 were corroborated with significantly higher photosynthetic rate, pollen fertility, stigma receptivity, and the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). Further, several genes involved in the maintenance of Pi homeostasis (GPH) were differentially regulated. The study thus revealed a wide-spectrum influence of the mutation in NH787 that contributed towards its higher Pi use efficiency (PUE).

A Comprehensive Analysis of MicroRNAs Expressed in Susceptible and Resistant Rice Cultivars during Rhizoctonia solani AG1-IA Infection Causing Sheath Blight Disease.

MicroRNAs regulate plant responses to fungal infections and immunity. In this study, miRNAs were identified in six rice cultivars during a Rhizoctonia solani Kühn AG1-IA infection using a deep sequencing approach. Known and novel miRNAs were analyzed in these rice cultivars, and a set of fungal infection/immunity-associated miRNAs and target genes were quantified by reverse transcription (RT)-qPCR in six rice cultivars. Additionally, the relative expression of these miRNAs was analyzed in different time points of the infection, wild species of rice, and in response to different strains of R. solani. Osa-miR1320-5p showed preferential expression during the fungal infection in all the six rice genotypes, while Osa-miR156d, Osa-miR159b, Osa-miR820c, and Osa-miR1876 were differentially regulated in susceptible and resistant genotypes. A greater degree of downregulation of miRNAs was observed during the initial time points of infection (24-72 h), suggesting a maximum molecular activity of rice-R. solani interaction and resistance response of the host during the early phase of infection. After R. solani infection, the expression of Osa-miR820c and Osa-miR156d was downregulated in Oryza rufipogon, O. alta, O. latifolia, and O. minuta, while Osa-miR397b was downregulated in all the wild rice species except O. officinalis. This study provided comprehensive information on the repertoire of miRNAs expressed in six sheath blight disease-susceptible and resistant indica and aus rice cultivars.

A Comprehensive Gene Expression Profile of Pectin Degradation Enzymes Reveals the Molecular Events during Cell Wall Degradation and Pathogenesis of Rice Sheath Blight Pathogen Rhizoctonia solani AG1-IA.

Sheath blight disease of rice caused by Rhizoctonia solani Kühn (teleomorph: Thanatephorus cucumeris) remains a global challenge due to the absence of reliable resistance genes and poor understanding of pathogen biology. Pectin, one of the most vital constituents of the plant cell wall, is targeted by pectin methylesterases, polygalacturonases, and few other enzymes of fungal pathogens. In this study, we catalogued the expressed genes of the fungal genome from RNAseq of R. solani infected four rice genotypes. Analysis of RNAseq revealed 3325 pathogen genes commonly expressed in all rice genotypes, in which 49, 490, and 83 genes were specific to BPT5204, Tetep, and Pankaj genotypes, respectively. To identify the early and late responding genes of R. solani during plant cell wall degradation, a real-time PCR analysis of 30 pectinolytic enzymes was done at six different time points after inoculation. The majority of these genes showed maximum induction at the 72 h time point, suggesting that it is the most crucial stage of infection. Pankaj showed lesser induction of these genes as compared to other genotypes. Leaf-blade tissue and 45 days old-growth stage are more favorable for the expression of pectin degradation genes of R. solani. Additionally, the expression analysis of these genes from four different strains of R. solani suggested differential regulation of genes but no distinct expression pattern between highly virulent and mild strains. The implications of the differential regulation of these genes in disease development have been discussed. This study provides the first such comprehensive analysis of R. solani genes encoding pectin degrading enzymes, which would help to decipher the pathogen biology and sheath blight disease development.

Genome-wide transcriptome profile of rice hybrids with and without Oryza rufipogon introgression reveals candidate genes for yield.

In this study, we compared genome-wide transcriptome profile of two rice hybrids, one with (test hybrid IR79156A/IL50-13) and the other without (control hybrid IR79156A/KMR3) O. rufipogon introgressions to identify candidate genes related to grain yield in the test hybrid. IL50-13 (Chinsurah Nona2 IET21943) the male parent (restorer) used in the test hybrid, is an elite BC4F8 introgression line of KMR3 with O. rufipogon introgressions. We identified 2798 differentially expressed genes (DEGs) in flag leaf and 3706 DEGs in panicle. Overall, 78 DEGs were within the major yield QTL qyld2.1 and 25 within minor QTL qyld8.2. The DEGs were significantly (p < 0.05) enriched in starch synthesis, phenyl propanoid pathway, ubiquitin degradation and phytohormone related pathways in test hybrid compared to control hybrid. Sequence analysis of 136 DEGs from KMR3 and IL50-13 revealed 19 DEGs with SNP/InDel variations. Of the 19 DEGs only 6 showed both SNP and InDel variations in exon regions. Of these, two DEGs within qyld2.1, Phenylalanine ammonia- lyase (PAL) (Os02t0626400-01, OsPAL2) showed 184 SNPs and 11 InDel variations and Similar to phenylalanine ammonia- lyase (Os02t0627100-01, OsPAL4) showed 205 SNPs and 13 InDel variations. Both PAL genes within qyld2.1 and derived from O. rufipogon are high priority candidate genes for increasing grain yield in rice.

Nucleocytoplasmic Trafficking of the Arabidopsis WD40 Repeat Protein XIW1 Regulates ABI5 Stability and Abscisic Acid Responses.

WD40 repeat-containing proteins (WD40 proteins) serve as versatile scaffolds for protein-protein interactions, modulating a variety of cellular processes such as plant stress and hormone responses. Here we report the identification of a WD40 protein, XIW1 (for XPO1-interacting WD40 protein 1), which positively regulates the abscisic acid (ABA) response in Arabidopsis. XIW1 is located in the cytoplasm and nucleus. We found that it interacts with the nuclear transport receptor XPO1 and is exported by XPO1 from the nucleus. Mutation of XIW1 reduces the induction of ABA-responsive genes and the accumulation of ABA Insensitive 5 (ABI5), causing mutant plants with ABA-insensitive phenotypes during seed germination and seedling growth, and decreased drought stress resistance. ABA treatment upregulates the expression of XIW1, and both ABA and abiotic stresses promote XIW1 accumulation in the nucleus, where it interacts with ABI5. Loss of XIW1 function results in rapid proteasomal degradation of ABI5. Taken together, these findings suggest that XIW1 is a nucleocytoplasmic shuttling protein and plays a positive role in ABA responses by interacting with and maintaining the stability of ABI5 in the nucleus.

CRISPR mediated genome engineering to develop climate smart rice: Challenges and opportunities.

Rice is a staple food crop, which ensures the calorie requirement of half of the world's population. With the continued increase in population, rice will play a key role in achieving the food security. However, in the constantly shrinking scenario of rice fields, the necessity of these extra grains of rice must be met by reducing the yield loss due to various abiotic and biotic stresses. The adverse effects of climate impact both quality and quantity of rice production. One of the most desirable applications of CRISPR/Cas technology would be to develop climate smart rice crop to sustain and enhance its productivity in the changing environment. In this review, we analyze the desirable phenotypes and responsible genetic factors, which can be utilized to develop tolerance against major abiotic stresses imposed by climate change through genome engineering. The possibility of utilizing the information from wild resources to engineer the corresponding alleles of cultivated rice has been presented. We have also shed light on available resources for generating genome edited rice lines. The CRISPR/Cas mediated genome editing strategies for engineering of novel genes were proposed to create a plant phenotype, which can face the adversities of climate change. Further, challenges of off-targets and undesirable phenotype were discussed.

Pectin induced transcriptome of a Rhizoctonia solani strain causing sheath blight disease in rice reveals insights on key genes and RNAi machinery for development of pathogen derived resistance.

KEY MESSAGE: RNAi mediated silencing of pectin degrading enzyme of R. solani gives a high level of resistance against sheath blight disease of rice. Rice sheath blight disease caused by Rhizoctonia solani Kuhn (telemorph; Thanatephorus cucumeris) is one of the most devastating fungal diseases which cause severe loss to rice grain production. In the absence of resistant cultivars, the disease is currently managed through fungicides which add to environmental pollution. To explore the potential of utilizing RNA interference (RNAi)-mediated resistance against sheath blight disease, we identified genes encoding proteins and enzymes involved in the RNAi pathway in this fungal pathogen. The RNAi target genes were deciphered by RNAseq analysis of a highly virulent strain of the R. solani grown in pectin medium. Additionally, pectin metabolism associated genes of R. solani were analyzed through transcriptome sequencing of infected rice tissues obtained from six diverse rice cultivars. One of the key candidate gene AG1IA_04727 encoding polygalacturonase (PG), which was observed to be significantly upregulated during infection, was targeted through RNAi to develop disease resistance. Stable expression of PG-RNAi construct in rice showed efficient silencing of AG1IA_04727 and suppression of sheath blight disease. This study highlights important information about the existence of RNAi machinery and key genes of R. solani which can be targeted through RNAi to develop pathogen-derived resistance, thus opening an alternative strategy for developing sheath blight-resistant rice cultivars.

Physical interaction of RTBV ORFI with D1 protein of Oryza sativa and Fe/Zn homeostasis play a key role in symptoms development during rice tungro disease to facilitate the insect mediated virus transmission.

Rice tungro disease is caused by the combined action of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). The RTBV is involved in the development of symptoms while RTSV is essential for virus transmission. We attempted to study the mode of action of RTBV in the development of symptoms. The tungro disease symptoms were attributed to viral interference in chlorophyll and carotenoids biosynthesis, photosynthesis machinery, iron/zinc homeostasis, and the genes encoding the enzymes associated with these biological processes of rice. The adverse effects of virus infection in photosystem II (PSII) activity was demonstrated by analyzing the Fv/Fm ratio, expression of psbA and cab1R genes, and direct interaction of RTBV ORF I protein with the D1 protein of rice. Since ORF I function is not yet known in the RTBV life cycle, this is the first report showing its involvement in regulating host photosynthesis process and symptoms development.

New breeding technique "genome editing" for crop improvement: applications, potentials and challenges.

Crop improvement is a continuous process in agriculture which ensures ample supply of food, fodder and fiber to burgeoning world population. Despite tremendous success in plant breeding and transgenesis to improve the yield-related traits, there have been several limitations primarily with the specificity in genetic modifications and incompatibility of host species. Because of this, new breeding techniques (NBTs) are gaining worldwide attention for crop improvement programs. Among the NBTs, genome editing (GE) using site-directed nucleases (SDNs) is an important and potential technique that overcomes limitations associated with classical breeding and transgenesis. These SDNs specifically target a compatible region in the gene/genome. The meganucleases (MgN), zinc finger nucleases (ZFN), transcription activator-like effectors nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated endonuclease (Cas) are being successfully employed for GE. These can be used for desired or targeted modifications of the native endogenous gene(s) or targeted insertion of cis/trans elements in the genomes of recipient organisms. Applications of these techniques appear to be endless ever since their discovery and several modifications in original technologies have further brought precision and accuracy in these methods. In this review, we present an overview of GE using SDNs with an emphasis on CRISPR/Cas system, their advantages, limitations and also practical considerations while designing experiments have been discussed. The review also emphasizes on the possible applications of CRISPR for improving economic traits in crop plants.

Proteomic and transcriptomic approaches to identify resistance and susceptibility related proteins in contrasting rice genotypes infected with fungal pathogen Rhizoctonia solani.

The devastating sheath blight disease caused by Rhizoctonia solani Kuhn (teleomorph: Thanatephorus cucumeris) causes major yield loss in most rice growing regions of the world. In this study, two moderately tolerant and four susceptible genotypes of rice were selected for R. solani induced proteome analysis using two-dimensional polyacrylamide gel electrophoresis. Forty five differentially expressed proteins (DEPs) were identified and analyzed by Mass Spectrometry. Based on their functions, these proteins were classified into different groups, viz., photosynthesis, resistance and pathogenesis, stress, cell wall metabolism and cytoskeleton development associated proteins, and hypothetical or uncharacterized proteins. Expression of 14 genes encoding DEPs was analyzed by quantitative PCR which showed consistency in transcripts and genes expression pattern. Furthermore, the expression of 16 other genes involved in diverse biological functions was analyzed. Up-regulation of these genes in the tolerant genotype Pankaj during sheath blight disease suggested efficient genetic regulation of this cultivar under stress. Also, expression analysis of conserved microRNAs (miRNAs) and their target genes revealed important role of miRNAs in post-transcriptional gene regulation during development of rice sheath blight disease. Genome-wide discovery of miRNAs and further characterization of DEPs and genes will help in better understanding of the molecular events during sheath blight disease development in rice.

Trait and Marker Associations in Oryza nivara and O. rufipogon Derived Rice Lines under Two Different Heat Stress Conditions.

Wild species and derived introgression lines (ILs) are a good source of genes for improving complex traits such as heat tolerance. The effect of heat stress on 18 yield traits was studied in four treatments in two seasons, under field conditions by subjecting 37 ILs and recurrent parents Swarna and KMR3, N22 mutants, and wild type and 2 improved rice cultivars to heat stress using polycover house method in wet season and late sowing method in dry season. Normal grown unstressed plants were controls. Both correlation and path coefficient analysis showed that the major contributing traits for high yield per plant (YPP) under heat stress conditions were tiller number, secondary branches in panicle, filled grain number, and percent spikelet fertility. Three ILs, K-377-24, K-16-3, and S-148 which gave the highest YPP of 12.30-32.52 g under heat stress in both the seasons were considered the most heat tolerant. In contrast, K-363-12, S-75, and Vandana which gave the least YPP of 5.36-10.84 g were considered heat susceptible. These lines are a good genetic resource for basic and applied studies on heat tolerance in rice. Genotyping using 49 SSR markers and single marker analysis (SMA) revealed 613 significant marker- trait associations in all four treatments. Significantly, nine markers (RM243, RM517, RM225, RM518, RM525, RM195, RM282, RM489, and RM570) on chromosomes 1, 2, 3, 4, 6, and 8 showed association with six traits (flag leaf spad, flag leaf thickness, vegetative leaf temperature, plant height, panicle number, and tiller number) under heat stress conditions in both wet and dry seasons. Genes such as heat shock protein binding DnaJ, Hsp70, and temperature-induced lipocalin-2 OsTIL-2 close to these markers are candidates for expression studies and evaluation for use in marker assisted selection for heat tolerance.