Integrated transcriptomics and metabolomics analysis provide insight into the resistance response of rice against brown planthopper

IntroductionThe brown planthopper (Nilaparvata lugens Stål, BPH) is one of the most economically significant pests of rice. The Bph30 gene has been successfully cloned and conferred rice with broad-spectrum resistance to BPH. However, the molecular mechanisms by which Bph30 enhances resistance to BPH remain poorly understood.MethodsHere, we conducted a transcriptomic and metabolomic analysis of Bph30-transgenic (BPH30T) and BPH-susceptible Nipponbare plants to elucidate the response of Bph30 to BPH infestation.ResultsTranscriptomic analyses revealed that the pathway of plant hormone signal transduction enriched exclusively in Nipponbare, and the greatest number of differentially expressed genes (DEGs) were involved in indole 3-acetic acid (IAA) signal transduction. Analysis of differentially accumulated metabolites (DAMs) revealed that DAMs involved in the amino acids and derivatives category were down-regulated in BPH30T plants following BPH feeding, and the great majority of DAMs in flavonoids category displayed the trend of increasing in BPH30T plants; the opposite pattern was observed in Nipponbare plants. Combined transcriptomics and metabolomics analysis revealed that the pathways of amino acids biosynthesis, plant hormone signal transduction, phenylpropanoid biosynthesis and flavonoid biosynthesis were enriched. The content of IAA significantly decreased in BPH30T plants following BPH feeding, and the content of IAA remained unchanged in Nipponbare. The exogenous application of IAA weakened the BPH resistance conferred by Bph30.DiscussionOur results indicated that Bph30 might coordinate the movement of primary and secondary metabolites and hormones in plants via the shikimate pathway to enhance the resistance of rice to BPH. Our results have important reference significance for the resistance mechanisms analysis and the efficient utilization of major BPH-resistance genes

Single-Cell RNA sequencing of leaf sheath cells reveals the mechanism of rice resistance to brown planthopper (Nilaparvata lugens)

The brown planthopper (BPH) (Nilaparvata lugens) sucks rice sap causing leaves to turn yellow and wither, often leading to reduced or zero yields. Rice co-evolved to resist damage by BPH. However, the molecular mechanisms, including the cells and tissues, involved in the resistance are still rarely reported. Single-cell sequencing technology allows us to analyze different cell types involved in BPH resistance. Here, using single-cell sequencing technology, we compared the response offered by the leaf sheaths of the susceptible (TN1) and resistant (YHY15) rice varieties to BPH (48 hours after infestation). We found that the 14,699 and 16,237 cells (identified via transcriptomics) in TN1 and YHY15 could be annotated using cell-specific marker genes into nine cell-type clusters. The two rice varieties showed significant differences in cell types (such as mestome sheath cells, guard cells, mesophyll cells, xylem cells, bulliform cells, and phloem cells) in the rice resistance mechanism to BPH. Further analysis revealed that although mesophyll, xylem, and phloem cells are involved in the BPH resistance response, the molecular mechanism used by each cell type is different. Mesophyll cell may regulate the expression of genes related to vanillin, capsaicin, and ROS production, phloem cell may regulate the cell wall extension related genes, and xylem cell may be involved in BPH resistance response by controlling the expression of chitin and pectin related genes. Thus, rice resistance to BPH is a complicated process involving multiple insect resistance factors. The results presented here will significantly promote the investigation of the molecular mechanisms underlying the resistance of rice to insects and accelerate the breeding of insect-resistant rice varieties

Comprehensive identification and characterization of lncRNAs and circRNAs reveal potential brown planthopper-responsive ceRNA networks in rice

Brown planthopper (Nilaparvata lugens Stål, BPH) is one of the most destructive pests of rice. Non-coding RNA plays an important regulatory role in various biological processes. However, comprehensive identification and characterization of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in BPH-infested rice have not been performed. Here, we performed a genome-wide analysis of lncRNAs and circRNAs in BPH6-transgenic (resistant, BPH6G) and Nipponbare (susceptible, NIP) rice plants before and after BPH feeding (early and late stage) via deep RNA-sequencing. A total of 310 lncRNAs and 129 circRNAs were found to be differentially expressed. To reveal the different responses of resistant and susceptible rice to BPH herbivory, the potential functions of these lncRNAs and circRNAs as competitive endogenous RNAs (ceRNAs) were predicted and investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Dual-luciferase reporter assays revealed that miR1846c and miR530 were targeted by the lncRNAs XLOC_042442 and XLOC_028297, respectively. In responsive to BPH infestation, 39 lncRNAs and 21 circRNAs were predicted to combine with 133 common miRNAs and compete for miRNA binding sites with 834 mRNAs. These mRNAs predictably participated in cell wall organization or biogenesis, developmental growth, single-organism cellular process, and the response to stress. This study comprehensively identified and characterized lncRNAs and circRNAs, and integrated their potential ceRNA functions, to reveal the rice BPH-resistance network. These results lay a foundation for further study on the functions of lncRNAs and circRNAs in the rice-BPH interaction, and enriched our understanding of the BPH-resistance response in rice

Development of Novel Rice Germplasm for Salt-Tolerance at Seedling Stage Using CRISPR-Cas9

Saline-alkali tolerant rice, as the first selected grain crop for improving coastal tidal land and saline-alkali land, has great potential for comprehensive utilization. In this study, an elite three-line restorer in breeding, R192, was taken as the receptor, and CRISPR/Cas9 technology was used to perform the directional editing of OsRR22, the main effect gene, which controls salt tolerance in rice. Eight transgenic plants of the T0 generation with the OsRR22 gene knockout were obtained, and the transgenic seedlings were screened by using PCR amplification and sequence comparison. The homozygous mutant lines, M16 and M18, with OsRR22 knockout, which did not contain a transgenic vector skeleton, were identified in the T2 generation. There were +1 bp and −20 bp in the exon regions of M16 and M18, respectively. At the three-leaf and one-heart stage, the seedlings were treated with 0.4% and 0.8% NaCl solution, and then their salt tolerance during the seedling stage was identified. The results showed that, without the salt treatment, no significant differences were found in plant height, the number of green leaves, total dry weight, and total fresh weight between the mutant lines M16 and M18 and the wildtype (WT) R192. However, after treatment with two different salt concentrations, the M16 and M18 mutants showed extremely significant differences in comparison with WT in plant height, the number of green leaves, total dry weight, and total fresh weight; between mutants and WT, there were significant differences in the number of green leaves, total fresh weight, and total dry weight after two salt treatments. Our results indicate the new germplasm with the OsRR22 mutation induced by CRISPR/Cas9 technology could improve the salt tolerance of rice, providing a reference for the improvement of salt tolerance of rice

Development of Novel Rice Germplasm for Salt-Tolerance at Seedling Stage Using CRISPR-Cas9

Saline-alkali tolerant rice, as the first selected grain crop for improving coastal tidal land and saline-alkali land, has great potential for comprehensive utilization. In this study, an elite three-line restorer in breeding, R192, was taken as the receptor, and CRISPR/Cas9 technology was used to perform the directional editing of OsRR22, the main effect gene, which controls salt tolerance in rice. Eight transgenic plants of the T0 generation with the OsRR22 gene knockout were obtained, and the transgenic seedlings were screened by using PCR amplification and sequence comparison. The homozygous mutant lines, M16 and M18, with OsRR22 knockout, which did not contain a transgenic vector skeleton, were identified in the T2 generation. There were +1 bp and −20 bp in the exon regions of M16 and M18, respectively. At the three-leaf and one-heart stage, the seedlings were treated with 0.4% and 0.8% NaCl solution, and then their salt tolerance during the seedling stage was identified. The results showed that, without the salt treatment, no significant differences were found in plant height, the number of green leaves, total dry weight, and total fresh weight between the mutant lines M16 and M18 and the wildtype (WT) R192. However, after treatment with two different salt concentrations, the M16 and M18 mutants showed extremely significant differences in comparison with WT in plant height, the number of green leaves, total dry weight, and total fresh weight; between mutants and WT, there were significant differences in the number of green leaves, total fresh weight, and total dry weight after two salt treatments. Our results indicate the new germplasm with the OsRR22 mutation induced by CRISPR/Cas9 technology could improve the salt tolerance of rice, providing a reference for the improvement of salt tolerance of rice

Genome-wide identification of long non-coding (lncRNA) in Nilaparvata lugens’s adaptability to resistant rice

Background The brown planthopper (BPH), Nilaparvata lugens (Stål), is a very destructive pest that poses a major threat to rice plants worldwide. BPH and rice have developed complex feeding and defense strategies in the long-term co-evolution. Methods To explore the molecular mechanism of BPH’s adaptation to resistant rice varieties, the lncRNA expression profiles of two virulent BPH populations were analyzed. The RNA-seq method was used to obtain the lncRNA expression data in TN1 and YHY15. Results In total, 3,112 highly reliable lncRNAs in TN1 and YHY15 were identified. Compared to the expression profiles between TN1 and YHY15, 157 differentially expressed lncRNAs, and 675 differentially expressed mRNAs were identified. Further analysis of the possible regulation relationships between differentially expressed lncRNAs and differentially expressed mRNAs, identified three pair antisense targets, nine pair cis-regulation targets, and 3,972 pair co-expressed targets. Function enriched found arginine and proline metabolism, glutathione metabolism, and carbon metabolism categories may significantly affect the adaptability in BPH when it is exposed to susceptible and resistant rice varieties. Altogether, it provided scientific data for the study of lncRNA regulation of brown planthopper resistance to rice. These results are helpful in the development of new control strategies for host defense against BPH and breeding rice for high yield

Transcriptome Analysis Revealed the Dynamic and Rapid Transcriptional Reprogramming Involved in Cold Stress and Related Core Genes in the Rice Seedling Stage

Cold damage is one of the most important environmental factors influencing crop growth, development, and production. In this study, we generated a pair of near-isogenic lines (NILs), Towada and ZL31, and Towada showed more cold sensitivity than ZL31 in the rice seedling stage. To explore the transcriptional regulation mechanism and the reason for phenotypic divergence of the two lines in response to cold stress, an in-depth comparative transcriptome study under cold stress was carried out. Our analysis uncovered that rapid and high-amplitude transcriptional reprogramming occurred in the early stage of cold treatment. GO enrichment and KEGG pathway analysis indicated that genes of the response to stress, environmental adaptation, signal transduction, metabolism, photosynthesis, and the MAPK signaling pathway might form the main part of the engine for transcriptional reprogramming in response to cold stress. Furthermore, we identified four core genes, OsWRKY24, OsCAT2, OsJAZ9, and OsRR6, that were potential candidates affecting the cold sensitivity of Towada and ZL31. Genome re-sequencing analysis between the two lines revealed that only OsWRKY24 contained sequence variations which may change its transcript abundance. Our study not only provides novel insights into the cold-related transcriptional reprogramming process, but also highlights the potential candidates involved in cold stress