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.

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

Introduction: The 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. Methods: Here, 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. Results: Transcriptomic 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. Discussion: Our 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.

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.

MUS81 is required for atypical recombination intermediate resolution but not crossover designation in rice.

The endonuclease methyl methanesulfonate and UV-sensitive protein 81 (MUS81) has been reported to participate in DNA repair during mitosis and meiosis. However, the exact meiotic function of MUS81 in rice remains unclear. Here, we use a combination of physiological, cytological, and genetic approaches to provide evidence that MUS81 functions in atypical recombination intermediate resolution rather than crossover designation in rice. Cytological and genetic analysis revealed that the total chiasma numbers in mus81 mutants were indistinguishable from wild-type. The numbers of HEI10 foci (the sites of interference-sensitive crossovers) in mus81 were also similar to that of wild-type. Moreover, disruption of MUS81 in msh5 or msh4 msh5 background did not further decrease chiasmata frequency, suggesting that rice MUS81 did not function in crossover designation. Mutation of FANCM and ZEP1 could enhance recombination frequency. Unexpectedly, chromosome fragments and bridges were frequently observed in mus81 zep1 and mus81 fancm, illustrating that MUS81 may resolve atypical recombination intermediates. Taken together, our data suggest that MUS81 contributes little to crossover designation but plays a crucial role in the resolution of atypical meiotic intermediates by working together with other anti-crossover factors.

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.

Improvement of Bacterial Blight Resistance in Two Conventionally Cultivated Rice Varieties by Editing the Noncoding Region.

xa13 is a recessive pleiotropic gene that positively regulates rice disease resistance and negatively regulates rice fertility; thus, seriously restricting its rice breeding application. In this study, CRISPR/Cas9 gene-editing technology was used to delete the Xa13 gene promoter partial sequence, including the pathogenic bacteria-inducible expression element. Rice with the edited promoter region lost the ability for pathogen-induced gene expression without affecting background gene expression in leaves and anthers, resulting in disease resistance and normal yield. The study also screened a family of disease-resistant and normal fertile plants in which the target sequence was deleted and the exogenous transgene fragment isolated in the T1 generation (transgene-free line). Important agronomic traits of the T2 generation rice were examined. T2 generation rice with/without exogenous DNA showed no statistical differences compared to the wild type in heading stage, plant height, panicles per plant, panicle length, or seed setting rate in the field. Two important conventional rice varieties, namely Kongyu131 (KY131, Geng/japonica) and Huanghuazhan (HHZ, Xian/indica), were successfully transformed, and disease-resistant and fertile materials were obtained. Currently, these are the two important conventional rice varieties in China that can be used directly for production after improvement. Expression of the Xa13 gene in the leaves of transgenic rice (KY-PD and HHZ-PD) was not induced after pathogen infection, indicating that this method can be used universally and effectively to promote the practical application of xa13, a recessive disease-resistant pleiotropic gene, for rice bacterial blight resistance. Our study on the regulation of gene expression by editing noncoding regions of the genes provides a new idea for the development of molecular design breeding in the future.

Molecular identification and efficacy assessment of a glufosinate-tolerant and brown planthopper-resistant transgenic rice line.

Insect pests and weeds are the two major biotic factors affecting crop yield in the modern agricultural system. In this study, a brown planthopper (BPH) resistance gene (BPH9) and glufosinate tolerance gene (bar) were stacked into a single T-DNA cassette and transformed into an indica rice (Oryza sativa L.) line Guangzhan 63-4S. A stable transgenic line H23 with a single T-DNA insert was generated, with the T-DNA cassette located on chromosome 3. Field resistance trial using H23 revealed high tolerance to glufosinate and excellent resistance to BPH. These results propose H23 as valuable germplasm for BPH-resistance and glufosinate-tolerance breeding in rice.

Identification and verification of grain shape QTLs by SNP array in rice.

Grain shape strongly influences the economic value and grain yield of rice. Thus, identifying quantitative trait loci (QTLs) for grain shape has been a longstanding goal in rice genetic research and breeding programs. Single nucleotide polymorphism (SNP) markers are ubiquitous in the rice genome and are more abundant and evenly distributed on the 12 rice chromosomes than traditional markers. An F2 population was genotyped using the RICE6K SNP array to elucidate the mechanisms governing grain shape. Thirty-five QTLs for grain shape were detected on 11 of 12 chromosomes over 2 years. The major QTL cluster qGS7 was detected in both years and displayed strong genetic effects on grain length and width, showing consistency with GL7/GW7. Some minor QTLs were also detected, and the effects of four QTLs on seed size were then validated using BC1F6 populations with residual heterozygous lines in each QTL region. Our findings provide insights into the molecular basis of grain shape as well as additional resources and approaches for producing hybrid high-yield rice varieties.

A Genome Doubling Event Reshapes Rice Morphology and Products by Modulating Chromatin Signatures and Gene Expression Profiling.

Evolutionarily, polyploidy represents a smart method for adjusting agronomically important in crops through impacts on genomic abundance and chromatin condensation. Autopolyploids have a relatively concise genetic background with great diversity and provide an ideal system to understand genetic and epigenetic mechanisms attributed to the genome-dosage effect. However, whether and how genome duplication events during autopolyploidization impact chromatin signatures are less understood in crops. To address it, we generated an autotetraploid rice line from a diploid progenitor, Oryza sativa ssp. indica 93-11. Using transposase-accessible chromatin sequencing, we found that autopolyploids lead to a higher number of accessible chromatin regions (ACRs) in euchromatin, most of which encode protein-coding genes. As expected, the profiling of ACR densities supported that the effect of ACRs on transcriptional gene activities relies on their positions in the rice genome, regardless of genome doubling. However, we noticed that genome duplication favors genic ACRs as the main drivers of transcriptional changes. In addition, we probed intricate crosstalk among various kinds of epigenetic marks and expression patterns of ACR-associated gene expression in both diploid and autotetraploid rice plants by integrating multiple-omics analyses, including chromatin immunoprecipitation sequencing and RNA-seq. Our data suggested that the combination of H3K36me2 and H3K36me3 may be associated with dynamic perturbation of ACRs introduced by autopolyploidization. As a consequence, we found that numerous metabolites were stimulated by genome doubling. Collectively, our findings suggest that autotetraploids reshape rice morphology and products by modulating chromatin signatures and transcriptional profiling, resulting in a pragmatic means of crop genetic improvement.

Characterization and comparative profiling of the small RNA transcriptomes in the Hemipteran insect Nilaparvata lugens.

MicroRNAs (miRNAs) are a group of small RNAs involved in various biological processes through negative regulation of mRNAs at the post-transcriptional level. The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most serious and destructive insect pests of rice. In the present study, two small RNA libraries of virulent N. lugens populations (Biotype I survives on susceptive rice variety TN1 and Biotype Y survives on moderately resistant rice variety YHY15) were constructed and sequenced using the high-throughput sequencing technology in order to identify the relationship between miRNAs of N.lugens and adaptation of BPH pests to rice resistance. In total 15,758,632 and 11,442,592 reads, corresponding to 3,144,026 and 2,550,049 unique sequences, were obtained in the two libraries (BPH-TN1 and BPH-YHY15 libraries), respectively. A total of 41 potential novel miRNAs were predicted in the two libraries, and 26 miRNAs showed significantly differential expression between two libraries. All miRNAs were significantly up-regulated in the BPH-TN1 library. Target genes likely regulated by these differentially expressed miRNAs were predicted using computational prediction. The functional annotation of target genes performed by Gene Ontology enrichment (GO) and Kyoto Encyclopedia of Genes and Genomes pathway analysis (KEGG) indicated that a majority of differential miRNAs were involved in "Metabolism" pathway. These results provided an understanding of the role of miRNAs in BPH to adaptability of BPH on rice resistance, and will be useful in developing new control strategies for host defense against BPH.

Development and validation of a PCR-based functional marker system for the brown planthopper resistance gene Bph14 in rice.

Brown planthopper (BPH) is the most damaging rice pest globally. Resistant varieties are the most effective and environmental strategy for protecting the rice crop from BPH. Functional markers (FMs) designed from polymorphic sites within gene sequences affecting phenotypic variation are highly efficient when used for marker assisted selection (MAS). Bph14 is the first and only cloned insect resistance gene so far in rice. Compared to the sequences of its non-effective alleles there are a number SNP differences. In this study, the method of allele-specific amplification (ASA) was adopted to design a simple, co-dominant, functional marker Bph14P/N for Bph14. Bph14P/N was combined with two specific dominant markers: one, named Bph14P, targets the promoter region of Bph14 and amplifies 566 bp fragments; and the other, Bph14N, targets the LRR region of bph14 and amplifies 345 bp fragments. Specificity and applicability of the functional marker system were verified in two breeding populations and a Chinese mini core collection of Oryza sativa. We recommend the use of this simple, low-cost marker system in routine genotyping for Bph14 in breeding populations.

Inheritance and expression of copies of transgenes 1Dx5 and 1Ax1 in elite wheat (Triticum aestivum L.) varieties transferred from transgenic wheat through conventional crossing.

To study the inheritance and expression of multiple copies of transgenes from transgenic wheat lines, three crosses between transgenic wheat lines B72-8-11b and B102-1-2 and Chinese elite wheat varieties Chuan89-107 and Emai18 were carried out. Chuan89-107x72-8-11b, Chuan89-107x102-1-2 and Emai18x72-8-11b, and F(1) plants were selfed or backcrossed to obtain different generation populations. Protein analysis in grains of F(1) and F(2) and backcross progenies of BC(1)F(1), BC(1)F(2), BC(1)F(3), BC(2)F(1), BC(2)F(2) and BC(2)F(3) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the transgenes 1Dx5 and 1Ax1 were expressed and segregated in the target wheat according to Mendelian laws. A range of 1Dx5 expression levels were observed in the progenies of Chuan89-107x72-8-11b and Emai18x72-8-11b, but the expression levels of 1Ax1 in progenies of Chuan89-107x102-1-2 rarely changed. It suggested that the two foreign genes had different mechanisms of expression in the cross progeny, even though they were produced in the same way and the foreign 1Dx5 gene of 5-10 copies had the more complicated expression mechanism than the 1Ax1 gene of 4-5 copies.