Molecular Approaches for Insect Pest Management in Rice

AbstractThis chapter focuses on the progress made in using molecular tools in understanding resistance in rice to insect pests and breeding rice for multiple and durable insect resistance. Currently, molecular markers are being extensively used to tag, map, introgress, and clone plant resistance genes against gall midge, planthoppers, and leafhoppers. Studies on cloned insect resistance genes are leading to a better understanding of plant defense against insect pests under different feeding guilds. While marker-assisted breeding is successfully tackling problems in durable and multiple pest resistance in rice, genomics of plants and insects has identified RNAi-based gene silencing as an alternative approach for conferring insect resistance. The use of these techniques in rice is in the developmental stage, with the main focus on brown planthopper and yellow stem borer. CRISPR-based genome editing techniques for pest control in plants has just begun. Insect susceptibility genes (negative regulators of resistance genes) in plants are apt targets for this approach while gene drive in insect populations, as a tool to study rice-pest interactions, is another concept being tested. Transformation of crop plants with diverse insecticidal genes is a proven technology with potential for commercial success. Despite advances in the development and testing of transgenic rice for insect resistance, no insect-resistant rice cultivar is now being commercially cultivated. An array of molecular tools is being used to study insect-rice interactions at transcriptome, proteome, metabolome, mitogenome, and metagenome levels, especially with reference to BPH and gall midge, and such studies are uncovering new approaches for insect pest management and for understanding population genetics and phylogeography of rice pests. Thus, it is evident that the new knowledge being gained through these studies has provided us with new tools and information for facing future challenges. However, what is also evident is that our attempts to manage rice pests cannot be a one-time effort but must be a continuing one

Expression Analysis of Novel microRNAs in Rice During High Temperature Stress

MicroRNAs (miRNAs) are small non-coding RNAs which play an important role in regulating the genes involved in plant growth and development. Several studies showed that miRNAs are involved in plants response to different kinds of abiotic stresses also. In our previous study, temperature responsive miRNAs were predicted in O.sativa. 27 miRNAs were predicted to be novel in rice using homology search. In continuation to our previous study, expression of 14 novel miRNAs was done in shoot and root of 13 days old seedlings of five different rice cultivars using real time PCR. Expression these miRNAs was analyzed in control and high temperature stress environment. Out of 14 predicted novel miRNAs, two novel miRNAs- miR157a and miR165a showed expression in all five rice cultivars. Interestingly, miR165a showed a differential expression pattern among heat tolerant (N22, IR64 and Rasi) and susceptible (Vandana and Sampada) cultivars suggesting that it might have specific role in high temperature tolerance

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

Expression Dynamics of Genes and microRNAs at Different Growth Stages and Heat Treatments in Contrasting High Temperature Responsive Rice Genotypes

The global warming-driven climate change is becoming a major challenge for rice cultivation in Asia and Africa. High-temperature stress impairs the physiology and growth of rice plant, and ultimately results in reduced grain yield. This study was aimed to decipher the physiological and molecular changes occurring during different growth stages of heat-tolerant (N22) and -susceptible (Vandana) rice cultivars under three different heat treatments. Chlorophyll content, membrane integrity, gas exchange parameters and expression of genes and miRNAs were analyzed in N22 and Vandana at seedling, vegetative, and reproductive growth stages after exposing to short and long duration of high temperature stress, and recovery. A number of genes and miRNAs showed dynamic changes in their expression patterns at different growth stages and heat treatments, highlighting the necessity to understand gene regulation before employing the genes for modification through transgenic or gene editing approaches. Predominantly N22 showed distinct and unique capability to reprogram its physiological and molecular machinery during prolonged heat stress at reproductive stage, suggesting that the dynamics in gene regulation is crucial to determine its heat tolerant ability. The study has larger implications in deploying genes for the development of heat tolerant rice cultivars through breeding, transgenic, and genome editing approaches

A multi-dimensional approach from seed-to-seed to understand and improve heat stress tolerance in rice

In changing climatic conditions, stress caused by high temperature poses a serious threat to rice cultivation. Physiological, biochemical, and molecular analysis of rice cultivars revealed that Nagina22 (N22) shows lesser reduction in chlorophyll content, net photosynthetic rate, spikelet fertility and grain yield, but increased membrane thermal stability, antioxidant enzymes activity and transpiration rate (E) at high temperature. DREB, RAB, LEA, and genes associated with hormones signalling were induced during germination, while OsFd (an iron sulphur cluster binding protein) and CWIP (cell wall integrity protein) emerged as high priority candidate genes in seedling and reproductive stages. Their function is being analysed by transgene expression and CRISPR/Cas genome editing approaches. Field screening in polyhouse, late sowing and temperature gradient chamber for 20 morpho-physiological traits indicated the importance of both yield and spikelet fertility, and photosynthesis traits. N22 showed the least Heat Susceptibility Index (HSI) for yield/plant, spikelet fertility, flag leaf SPAD and stomatal conductance, while Vandana showed the highest HSI for spikelet fertility and flag leaf temperature. QTLs for HSI of spikelet fertility were identified on chromosome 1 and HSI of yield per plant on chromosomes 1, 2, 3, 4, 7 and 8; and PV of 6% to 57% using 174 F2-3 Vandana x N22 mapping population. Simultaneously, RNAseq was performed to identify the genome wide miRNAs and transcriptome of N22 and Vandana from shoot and root after short and long duration of heat stress treatments; and recovery phase for an eQTL-guided function-related co-expression analysis to identify the putative regulators and gene regulatory networks

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

Long non-coding RNA-mediated epigenetic response for abiotic stress tolerance in plants

Plants perceive environmental fluctuations as stress and confront several stresses throughout their life cycle individually or in combination. Plants have evolved their sensing and signaling mechanisms to perceive and respond to a variety of stresses. Epigenetic regulation plays a critical role in the regulation of genes, spatiotemporal expression of genes under stress conditions and imparts a stress memory to encounter future stress responses. It is quintessential to integrate our understanding of genetics and epigenetics to maintain plant fitness, achieve desired genetic gains with no trade-offs, and durable long-term stress tolerance. The long non-coding RNA >200 nts having no coding potential (or very low) play several roles in epigenetic memory, contributing to the regulation of gene expression and the maintenance of cellular identity which include chromatin remodeling, imprinting (dosage compensation), stable silencing, facilitating nuclear organization, regulation of enhancer-promoter interactions, response to environmental signals and epigenetic switching. The lncRNAs are involved in a myriad of stress responses by activation or repression of target genes and hence are potential candidates for deploying in climate-resilient breeding programs. This review puts forward the significant roles of long non-coding RNA as an epigenetic response during abiotic stresses in plants and the prospects of deploying lncRNAs for designing climate-resilient plants

Deep sequencing of small RNAs reveals ribosomal origin of microRNAs in Oryza sativa and their regulatory role in high temperature

MicroRNAs are small noncoding regulatory RNAs which control gene expression by mRNA degradation or translational repression. They are significant molecular players regulating important biological processes such as developmental timing and stress response. We report here the discovery of miRNAs derived from ribosomal DNA using the small RNA datasets of 16 deep sequencing libraries of rice. Twelve putative miRNAs were identified based on highly stringent criteria of novel miRNA prediction. Surprisingly, 10 putative miRNAs (mi_7403, mi_8435, mi_12675, mi_4266, mi_4758, mi_4218, mi_8200, mi_4644, mi_14291, mi_16235) originated from rDNA of rice chromosome 9. Expression analysis of putative miRNAs and their target genes in heat tolerant and susceptible rice cultivars in control and high temperature treated seedlings revealed differential regulation of rDNA derived miRNAs. This is the first report of rDNA derived miRNAs in rice which indicates their role in gene regulation during high temperature stress in plants. Further studies in this area will open new research challenges and opportunities to broaden our knowledge on gene regulation mechanisms

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. Abstract 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

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