Youlu Yuan

Analysis of sea-island cotton and upland cotton in response to Verticillium dahliae infection by RNA sequencing

BackgroundCotton Verticillium wilt is a serious soil-borne vascular disease that causes great economic loss each year. However, due to the lack of resistant varieties of upland cotton, the molecular mechanisms of resistance to this disease, especially to the pathogen Verticillium dahliae, remain unclear.ResultsWe used the RNA-seq method to research the molecular mechanisms of cotton defence responses to different races of Verticillium dahliae by comparing infected sea-island cotton and upland cotton. A total of 77,212 unigenes were obtained, and the unigenes were subjected to BLAST searching and annotated using the GO and KO databases. Six sets of digital gene expression data were mapped to the reference transcriptome. The gene expression profiles of cotton infected with Verticillium dahliae were compared to those of uninfected cotton; 44 differentially expressed genes were identified. Regarding genes involved in the phenylalanine metabolism pathway, the hydroxycinnamoyl transferase gene (HCT) was upregulated in upland cotton whereas PAL, 4CL, CAD, CCoAOMT, and COMT were upregulated in sea-island cotton. Almost no differentially expressed genes in this pathway were identified in sea-island cotton and upland cotton when they were infected with V. dahliae V991 and V. dahliae D07038, respectively.ConclusionsOur comprehensive gene expression data at the transcription level will help elucidate the molecular mechanisms of the cotton defence response to V. dahliae. By identifying the genes involved in the defence response of each type of cotton to V. dahliae, our data not only provide novel molecular information for researchers, but also help accelerate research on genes involved in defences in cotton.

Identification of novel microRNAs in the Verticillium wilt-resistant upland cotton variety KV-1 by high-throughput sequencing

Plant microRNAs (miRNAs) play essential roles in the post-transcriptional regulation of gene expression during development, flowering, plant growth, metabolism, and stress responses. Verticillium wilt is one of the vascular disease in plants, which is caused by the Verticillium dahlia and leads to yellowing, wilting, lodging, damage to the vascular tissue, and death in cotton plants. Upland cotton varieties KV-1 have shown resistance to Verticillium wilt in multiple levels. However, the knowledge regarding the post-transcriptional regulation of the resistance is limited. Here two novel small RNA (sRNA) libraries were constructed from the seedlings of upland cotton variety KV-1, which is highly resistant to Verticillium wilts and inoculated with the V991 and D07038 Verticillium dahliae (V. dahliae) of different virulence strains. Thirty-seven novel miRNAs were identified after sequencing these two libraries by the Illumina Solexa system. According to sequence homology analysis, potential target genes of these miRNAs were predicted. With no more than three sequence mismatches between the novel miRNAs and the potential target mRNAs, we predicted 49 target mRNAs for 24 of the novel miRNAs.These target mRNAs corresponded to genes were found to be involved in plant–pathogen interactions, endocytosis, the mitogen-activated protein kinase (MAPK) signaling pathway, and the biosynthesis of isoquinoline alkaloid, terpenoid backbone, primary bile acid and secondary metabolites. Our results showed that some of these miRNAs and their relative gene are involved in resistance to Verticillium wilts. The identification and characterization of miRNAs from upland cotton could help further studies on the miRNA regulatory mechanisms of resistance to Verticillium wilt.

Gene expression profiling during gland morphogenesis of a mutant and a glandless upland cotton

To identify genes involved in pigment gland morphogenesis in cotton, gene expression was profiled using genechip (Affymetrix) during pigment gland morphogenesis in cotton variety Xiangmian-18, which has glandless seeds but glanded plants, and a glandless line, N5. The results showed that 303 genes were differentially expressed by a factor greater than two during gland morphogenesis; 59% (180) of these genes shared similarity with known genes in GenBank. These genes play roles in defense response, response to oxidative stress, peroxidase activity, and other metabolic pathways. KOBAS (KEGG Orthology-Based Annotation System) indicate that these genes are involved in 68 biochemical pathways. These findings suggest that the related defense response, gossypol biosynthesis pathway and other complex regulation may be associated with pigment gland morphogenesis in cotton. The results may provide a basis for further study and serve as a guide for related research.

Characterization, Expression, and Functional Analysis of a Novel NAC Gene Associated with Resistance to Verticillium Wilt and Abiotic Stress in Cotton.

Elucidating the mechanism of resistance to biotic and abiotic stress is of great importance in cotton. In this study, a gene containing the NAC domain, designated GbNAC1, was identified from Gossypium barbadense L. Homologous sequence alignment indicated that GbNAC1 belongs to the TERN subgroup. GbNAC1 protein localized to the cell nucleus. GbNAC1 was expressed in roots, stems, and leaves, and was especially highly expressed in vascular bundles. Functional analysis showed that cotton resistance to Verticillium wilt was reduced when the GbNAC1 gene was silenced using the virus-induced gene silencing (VIGS) method. GbNAC1-overexpressing Arabidopsis showed enhanced resistance to Verticillium dahliae compared to wild-type. Thus, GbNAC1 is involved in the positive regulation of resistance to Verticillium wilt. In addition, analysis of GbNAC1-overexpressing Arabidopsis under different stress treatments indicated that it is involved in plant growth, development, and response to various abiotic stresses (ABA, mannitol, and NaCl). This suggests that GbNAC1 plays an important role in resistance to biotic and abiotic stresses in cotton. This study provides a foundation for further study of the function of NAC genes in cotton and other plants.

Genome-wide identification of the TIFY gene family in three cultivated Gossypium species and the expression of JAZ genes

TIFY proteins are plant-specific proteins containing TIFY, JAZ, PPD and ZML subfamilies. A total of 50, 54 and 28 members of the TIFY gene family in three cultivated cotton species—Gossypium hirsutum, Gossypium barbadense and Gossypium arboretum—were identified, respectively. The results of phylogenetic analysis showed that these TIFY genes were divided into eight clusters. The different clusters of gene family members often have similar gene structures, including the number of exons. The results of quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that different JAZ genes displayed distinct expression patterns in the leaves of upland cotton under treatment with Gibberellin (GA), methyl jasmonate (MeJA), Jasmonic acid (JA) and abscisic acid (ABA). Different groups of JAZ genes exhibited different expression patterns in cotton leaves infected with Verticillium dahliae. The results of the comparative analysis of TIFY genes in the three cultivated species will be useful for understanding the involvement of these genes in development and stress resistance in cotton.

Profiling Gene Expression During Gland Morphogenesis of a Glanded and a Glandless Upland Cotton

The pigment gland is an important character of the Gossypium plant. With the aim of identifying genes involved in pigment gland morphogenesis in cotton, gene expression during pigment gland morphogenesis in Chuan 2802, which is glanded both in seed and plant, and a glandless line N5 was profiled using Affymetrix Cotton microarray. The results showed that there were 564 differentially expressed genes greater than twofold during gland morphogenesis. About 60.2% of these genes shares similarity with known genes on GenBank and about 39.8% with no functional description in the database. These described genes may play roles in defense response, response to oxidative stress, peroxidase activity, and the other metabolic pathways. The KEGG Orthology-Based Annotation System indicated that these above twofold expressed genes involved seven biochemical pathways on KEGG. These findings suggest that a complicated regulation is associated with pigment gland morphogenesis and the associated defense response including gossypol biosynthesis in cotton.

Molecular characterisation and functional analysis of a cytochrome P450 gene in cotton

Abstract Verticillium wilt causes devastating loss of yield and quality in many crops, including cotton. To determine the molecular mechanism of resistance to verticillium wilt in cotton, we isolated a new cytochrome P450 gene, CYP94C1, and analysed its function. We obtained the complete open reading frame, which encodes a protein of 500 amino acids. The results of the functional analysis showed that resistance to verticillium wilt was enhanced when the gene was silenced using the virus-induced gene silencing (VIGS) method in cotton. GbCYP94Cl-overexpressing Arabidopsis was more susceptible to Verticillium dahliae compared with the wild-type. In addition, GbCYP94C1 played a role in plant wound responses and in the expression of certain jasmonic acid (JA) signalling pathway. The results suggest that GbCYP94C1 plays a role in verticillium wilt resistance in cotton via the JA signalling pathway.

MicroRNA expression profiling during upland cotton gland forming age by microarray and quantitative reverse-transcription polymerase chain reaction (qRT- PCR)

Plant microRNAs (miRNAs) have important impacts on growth, development, flowering, metabolism and response to stress. Studies indicate that post-transcriptional processes are important for regulating gene expression during development. However, we still have very limited knowledge on the regulatory mechanisms associated with this process. In particular, the function of miRNAs during gland morphogenesis in cotton remains unknown. In this study, we used the Affymetrix GeneChip miRNA Array (v11.0-ther Species) and quantitative reverse transcriptase-PCR (qRT-PCR) to identify additional microRNAs during gland morphogenesis of near-isogenic lines in upland cotton. The results showed that 30 miRNAs were differentially expressed: 24 up-regulated (miR156, miR157, miR166 and miR390 families) and six down-regulated (miR149, miR169, miR289, miR705, miR1224 and miR1227 families). Some microRNAs, such as ghb-miR169a_st and ghr-miR166b_st, were confirmed by qRT-PCR assays. There was no significant difference in miRNA levels between the microarray and qRT-PCR. Analysis of the transcript data for some miRNA target genes indicated that they play an important role in the pathogenesis and development of gland morphogenesis. In summary, our results showed that some known miRNAs were expressed in the gland of upland cotton, and most of them were of low abundance. This data may be useful in future studies associated with gland control involved in the terpenoid aldehyde biosynthesis pathway, genetic engineering and molecular breeding of cotton.

Changes in DNA methylation assessed by genomic bisulfite sequencing suggest a role for DNA methylation in cotton fruiting branch development

Cotton plant architecture, including fruit branch formation and flowering pattern, influences plant light exploitation, cotton yield and planting cost. DNA methylation has been widely observed at different developmental stages in both plants and animals and is associated with regulation of gene expression, chromatin remodelling, genome protection and other functions. Here, we investigated the global epigenetic reprogramming during the development of fruiting branches and floral buds at three developmental stages: the seedling stage, the pre-squaring stage and the squaring stage. We first identified 22 cotton genes which potentially encode DNA methyltransferases and demethylases. Among them, the homologous genes of CMT, DRM2 and MET1 were upregulated at pre-squaring and squaring stages, suggesting that DNA methylation is involved in the development of floral buds and fruit branches. Although the global methylation at all of three developmental stages was not changed, the CHG-type methylation of non-expressed genes was higher than those of expressed genes. In addition, we found that the expression of the homologous genes of the key circadian rhythm regulators, including CRY, LHY and CO, was associated with changes of DNA methylation at three developmental stages.

Identification of circularRNAs and their targets in Gossypium under Verticillium wilt stress based on RNA-seq

Circular RNAs (circRNAs), a class of recently discovered non-coding RNAs, play a role in biological and developmental processes. A recent study showed that circRNAs exist in plants and play a role in their environmental stress responses. However, cotton circRNAs and their role in Verticillium wilt response have not been identified up to now. In this study, two CSSLs (chromosome segment substitution lines) of G.barbadense introgressed into G. hirsutum, CSSL-1 and CSSL-4 (a resistant line and a susceptible line to Verticillium wilt, respectively), were inoculated with V. dahliae for RNA-seq library construction and circRNA analysis. A total of 686 novel circRNAs were identified. CSSL-1 and CSSL-4 had similar numbers of circRNAs and shared many circRNAs in common. However, CSSL-4 differentially expressed approximately twice as many circRNAs as CSSL-1, and the differential expression levels of the common circRNAs were generally higher in CSSL-1 than in CSSL-4. Moreover, two C-RRI comparisons, C-RRI-vs-C-RRM and C-RRI-vs-C-RSI, possessed a large proportion (approximately 50%) of the commonly and differentially expressed circRNAs. These results indicate that the differentially expressed circRNAs may play roles in the Verticillium wilt response in cotton. A total of 280 differentially expressed circRNAs were identified. A Gene Ontology analysis showed that most of the ‘stimulus response’ term source genes were NBS family genes, of which most were the source genes from the differentially expressed circRNAs, indicating that NBS genes may play a role in Verticillium wilt resistance and might be regulated by circRNAs in the disease-resistance process in cotton.