Strigolactones positively regulate Verticillium wilt resistance in cotton via crosstalk with other hormones.

Verticillium wilt caused by Verticillium dahliae is a serious vascular disease in cotton (Gossypium spp.). V. dahliae induces the expression of the CAROTENOID CLEAVAGE DIOXYGENASE 7 (GauCCD7) gene involved in strigolactone (SL) biosynthesis in Gossypium australe, suggesting a role for SLs in Verticillium wilt resistance. We found that the SL analog rac-GR24 enhanced while the SL biosynthesis inhibitor TIS108 decreased cotton resistance to Verticillium wilt. Knock-down of GbCCD7 and GbCCD8b genes in island cotton (Gossypium barbadense) decreased resistance, whereas overexpression of GbCCD8b in upland cotton (Gossypium hirsutum) increased resistance to Verticillium wilt. Additionally, Arabidopsis (Arabidopsis thaliana) SL mutants defective in CCD7 and CCD8 putative orthologs were susceptible, whereas both Arabidopsis GbCCD7- and GbCCD8b-overexpressing plants were more resistant to Verticillium wilt than wild-type (WT) plants. Transcriptome analyses showed that several genes related to the jasmonic acid (JA)- and abscisic acid (ABA)-signaling pathways, such as MYELOCYTOMATOSIS 2 (GbMYC2) and ABA-INSENSITIVE 5, respectively, were upregulated in the roots of WT cotton plants in responses to rac-GR24 and V. dahliae infection but downregulated in the roots of both GbCCD7- and GbCCD8b-silenced cotton plants. Furthermore, GbMYC2 suppressed the expression of GbCCD7 and GbCCD8b by binding to their promoters, which might regulate the homeostasis of SLs in cotton through a negative feedback loop. We also found that GbCCD7- and GbCCD8b-silenced cotton plants were impaired in V. dahliae-induced reactive oxygen species (ROS) accumulation. Taken together, our results suggest that SLs positively regulate cotton resistance to Verticillium wilt through crosstalk with the JA- and ABA-signaling pathways and by inducing ROS accumulation.

lncRNA7 and lncRNA2 modulate cell wall defense genes to regulate cotton resistance to Verticillium wilt.

In plants, long noncoding RNAs (lncRNAs) regulate disease resistance against fungi and other pathogens. However, the specific mechanism behind this regulation remains unclear. In this study, we identified disease resistance-related lncRNAs as well as their regulating genes and assessed their functions by infection of cotton (Gossypium) chromosome segment substitution lines with Verticillium dahliae. Our results demonstrated that lncRNA7 and its regulating gene Pectin methylesterase inhibitor 13 (GbPMEI13) positively regulated disease resistance via the silencing approach, while ectopic overexpression of GbPMEI13 in Arabidopsis (Arabidopsis thaliana) promoted growth and enhanced resistance to V. dahliae. In contrast, lncRNA2 and its regulating gene Polygalacturonase 12 (GbPG12) negatively regulated resistance to V. dahliae. We further found that fungal disease-related agents, including the pectin-derived oligogalacturonide (OG), could downregulate the expression of lncRNA2 and GbPG12, leading to pectin accumulation. Conversely, OG upregulated the expression of lncRNA7, which encodes a plant peptide phytosulfokine (PSK-α), which was confirmed by lncRNA7 overexpression and Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS) experiments. We showed that PSK-α promoted 3-Indoleacetic acid (IAA) accumulation and activated GbPMEI13 expression through Auxin Response Factor 5. Since it is an inhibitor of pectin methylesterase (PME), GbPMEI13 promotes pectin methylation and therefore increases the resistance to V. dahliae. Consistently, we also demonstrated that GbPMEI13 inhibits the mycelial growth and spore germination of V. dahliae in vitro. In this study, we demonstrated that lncRNA7, lncRNA2, and their regulating genes modulate cell wall defense against V. dahliae via auxin-mediated signaling, providing a strategy for cotton breeding.

The use of ribosome-nascent chain complex-seq to reveal the translated mRNA profile and the role of ASN1 in resistance to Verticillium wilt in cotton.

We combined traditional mRNA-seq and RNC-seq together to reveal post-transcriptional regulation events impacting gene expression and interactions between the serious fungal pathogen Verticillium dahliae and a susceptible host, Gossypium hirsutum TM-1. After screening the differentially expressed and translated genes, V. dahliae infection was observed to influence gene transcription and translation in its host. Interestingly, the asparagine synthase (ASN1) gene transcripts increased significantly with the increase of infection time, while the rate of ASN1 protein accumulation in host TM-1 was distinctly lower than that in resistant hosts. We knocked down the ASN1 gene in resistant plants (ZZM2), and found that Verticillium-resistance was significantly reduced upon knockdown of ASN1. Our study revealed both transcriptional and post-transcriptional regulation of gene expression in TM-1 cotton plants infected by V. dahliae, and showed that ASN1 functions in the V. dahliae resistance process. These insights support breeding of disease resistance in cotton.

Genome sequencing of the Australian wild diploid species Gossypium australe highlights disease resistance and delayed gland morphogenesis.

The diploid wild cotton species Gossypium australe possesses excellent traits including resistance to disease and delayed gland morphogenesis, and has been successfully used for distant breeding programmes to incorporate disease resistance traits into domesticated cotton. Here, we sequenced the G. australe genome by integrating PacBio, Illumina short read, BioNano (DLS) and Hi-C technologies, and acquired a high-quality reference genome with a contig N50 of 1.83 Mb and a scaffold N50 of 143.60 Mb. We found that 73.5% of the G. australe genome is composed of various repeat sequences, differing from those of G. arboreum (85.39%), G. hirsutum (69.86%) and G. barbadense (69.83%). The G. australe genome showed closer collinear relationships with the genome of G. arboreum than G. raimondii and has undergone less extensive genome reorganization than the G. arboreum genome. Selection signature and transcriptomics analyses implicated multiple genes in disease resistance responses, including GauCCD7 and GauCBP1, and experiments revealed induction of both genes by Verticillium dahliae and by the plant hormones strigolactone (GR24), salicylic acid (SA) and methyl jasmonate (MeJA). Experiments using a Verticillium-resistant domesticated G. barbadense cultivar confirmed that knockdown of the homologues of these genes caused a significant reduction in resistance against Verticillium dahliae. Moreover, knockdown of a newly identified gland-associated gene GauGRAS1 caused a glandless phenotype in partial tissues using G. australe. The G. australe genome represents a valuable resource for cotton research and distant relative breeding as well as for understanding the evolutionary history of crop genomes.

Functional analysis of the GbDWARF14 gene associated with branching development in cotton.

Plant architecture, including branching pattern, is an important agronomic trait of cotton crops. In recent years, strigolactones (SLs) have been considered important plant hormones that regulate branch development. In some species such as Arabidopsis, DWARF14 is an unconventional receptor that plays an important role in the SL signaling pathway. However, studies on SL receptors in cotton are still lacking. Here, we cloned and analysed the structure of the GbD14 gene in Gossypium barbadense and found that it contains the domains necessary for a SL receptor. The GbD14 gene was expressed primarily in the roots, leaves and vascular bundles, and the GbD14 protein was determined via GFP to localize to the cytoplasm and nucleus. Gene expression analysis revealed that the GbD14 gene not only responded to SL signals but also was differentially expressed between cotton plants whose types of branching differed. In particular, GbD14 was expressed mainly in the axillary buds of normal-branching cotton, while it was expressed the most in the leaves of nulliplex-branch cotton. In cotton, the GbD14 gene can be induced by SL and other plant hormones, such as indoleacetic acid, abscisic acid, and jasmonic acid. Compared with wild-type Arabidopsis, GbD14-overexpressing Arabidopsis responded more rapidly to SL signals. Moreover, we also found that GbD14 can rescue the multi-branched phenotype of Arabidopsis Atd14 mutants. Our results indicate that the function of GbD14 is similar to that of AtD14, and GbD14 may be a receptor for SL in cotton and involved in regulating branch development. This research provides a theoretical basis for a profound understanding of the molecular mechanism of branch development and ideal plant architecture for cotton breeding improvements.

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.

Genome-wide comparative analysis of NBS-encoding genes in four Gossypium species.

BACKGROUND: Nucleotide binding site (NBS) genes encode a large family of disease resistance (R) proteins in plants. The availability of genomic data of the two diploid cotton species, Gossypium arboreum and Gossypium raimondii, and the two allotetraploid cotton species, Gossypium hirsutum (TM-1) and Gossypium barbadense allow for a more comprehensive and systematic comparative study of NBS-encoding genes to elucidate the mechanisms of cotton disease resistance. RESULTS: Based on the genome assembly data, 246, 365, 588 and 682 NBS-encoding genes were identified in G. arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively. The distribution of NBS-encoding genes among the chromosomes was nonrandom and uneven, and was tended to form clusters. Gene structure analysis showed that G. arboreum and G. hirsutum possessed a greater proportion of CN, CNL, and N genes and a lower proportion of NL, TN and TNL genes compared to that of G. raimondii and G. barbadense, while the percentages of RN and RNL genes remained relatively unchanged. The percentage changes among them were largest for TNL genes, about 7 times. Exon statistics showed that the average exon numbers per NBS gene in G. raimondii and G. barbadense were all greater than that in G. arboretum and G. hirsutum. Phylogenetic analysis revealed that the TIR-NBS genes of G. barbadense were closely related with that of G. raimondii. Sequence similarity analysis showed that diploid cotton G. arboreum possessed a larger proportion of NBS-encoding genes similar to that of allotetraploid cotton G. hirsutum, while diploid G. raimondii possessed a larger proportion of NBS-encoding genes similar to that of allotetraploid cotton G. barbadense. The synteny analysis showed that more NBS genes in G. raimondii and G. arboreum were syntenic with that in G. barbadense and G. hirsutum, respectively. CONCLUSIONS: The structural architectures, amino acid sequence similarities and synteny of NBS-encoding genes between G. arboreum and G. hirsutum, and between G. raimondii and G. barbadense were the highest among comparisons between the diploid and allotetraploid genomes, indicating that G. hirsutum inherited more NBS-encoding genes from G. arboreum, while G. barbadense inherited more NBS-encoding genes from G. raimondii. This asymmetric evolution of NBS-encoding genes may help to explain why G. raimondii and G. barbadense are more resistant to Verticillium wilt, whereas G. arboreum and G. hirsutum are more susceptible to Verticillium wilt. The disease resistances of the allotetraploid cotton were related to their NBS-encoding genes especially in regard from which diploid progenitor they were derived, and the TNL genes may have a significant role in disease resistance to Verticillium wilt in G. raimondii and G. barbadense.

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.

To Be a Flower or Fruiting Branch: Insights Revealed by mRNA and Small RNA Transcriptomes from Different Cotton Developmental Stages.

The architecture of the cotton plant, including fruit branch formation and flowering pattern, is the most important characteristic that directly influences light exploitation, yield and cost of planting. Nulliplex branch is a useful phenotype to study cotton architecture. We used RNA sequencing to obtain mRNA and miRNA profiles from nulliplex- and normal-branch cotton at three developmental stages. The differentially expressed genes (DEGs) and miRNAs were identified that preferentially/specifically expressed in the pre-squaring stage, which is a key stage controlling the transition from vegetative to reproductive growth. The DEGs identified were primarily enriched in RNA, protein, and signalling categories in Gossypium barbadense and Gossypium hirsutum. Interestingly, during the pre-squaring stage, the DEGs were predominantly enriched in transcription factors in both G. barbadense and G. hirsutum, and these transcription factors were mainly involved in branching and flowering. Related miRNAs were also identified. The results showed that fruit branching in cotton is controlled by molecular pathways similar to those in Arabidopsis and that multiple regulated pathways may affect the development of floral buds. Our study showed that the development of fruit branches is closely related to flowering induction and provides insight into the molecular mechanisms of branch and flower development in cotton.