•The first proteomic study on cotton's responses against Rhizoctonia solani infection was conducted.•174 differentially accumulated proteins were identified based on iTRAQ technique.•Several cellular processes tightly associated with the disease protection were recognized.The soil-borne necrotrophic pathogen fungus Rhizoctonia solani is destructive, causing disease in various important crops. To date, little is known about the host defence mechanism in response to invasion of R. solani. Here, an iTRAQ-based proteomic analysis was employed to investigate pathogen-responsive proteins in the disease tolerant/resistant cotton cultivar CRI35. A total of 174 differentially accumulated proteins (DAPs) were identified after inoculation of cotton plants with R. solani. Functional categorization analysis indicated that these DAPs can be divided into 12 subclasses. Notably, a large portion of DAPs are known to function in reactive oxygen species (ROS) metabolism and the expression of several histone-modifying and DNA methylating proteins were significantly induced upon challenge with the fungus, indicating that the redox homeostasis and epigenetic regulation are important for cotton defence against the pathogen. Additionally, the expression of proteins involved in phenylpropanoid biosynthesis was markedly changed in response to pathogen invasion, which may reflect a particular contribution of secondary metabolism in protection against the fungal attack in cotton. Together, our results indicate that the defence response of cotton plants to R. solani infection is active and multifaceted and involves the induction of proteins from various innate immunity-related pathways.SignificanceCotton damping-off is a destructive disease caused by the necrotrophic fungus Rhizoctonia solani. To date, the host defence mechanism involved in the disease protection remains largely unknown. Here, we reported the first proteomic analysis on cotton immune responses against R. solani infection. Employing iTRAQ technique, we obtained a total of 174 differentially accumulated proteins (DAPs) that can be classified into 12 functional groups. Further analysis indicated that ROS homeostasis, epigenetic regulation and phenylpropanoid biosynthesis were tightly associated with the innate immune responses against R. solani infection in cotton. The obtained data provide not only important information for understanding the molecular mechanism involved in plant-R. solani interaction but also application clues for genetic breeding of crops with improved R. solani resistance.

Growing evidence indicates that actin cytoskeleton is involved in plant innate immune responses, but the functional mechanism remains largely unknown. Here, we investigated the behavior of a cotton profilin gene (GhPFN2) in response to Verticillium dahliae invasion, and evaluated its contribution to plant defense against this soil-borne fungal pathogen. GhPFN2 expression was up-regulated when cotton root was inoculated with V. dahliae, and the actin architecture was reorganized in the infected root cells, with a clear increase in the density of filamentous actin and the extent of actin bundling. Compared to the wild type, GhPFN2-overexpressing cotton plants showed enhanced protection against V. dahliae infection and the actin cytoskeleton organization in root epidermal cells was clearly altered, which phenocopied that of the wild-type (WT) root cells challenged with V. dahliae. These results provide a solid line of evidence showing that actin cytoskeleton reorganization involving GhPFN2 is important for defense against V. dahliae infection.

A comparative proteomic analysis was performed to identify the differences between brown cotton fiber and a white near-isogenic line, and 78 differential spots were identified at three time points (12-, 18-, and 24-day post-anthesis [DPA]) using MALDI-TOF/TOF. Our data illustrate several aspects of pigment synthesis and fiber development in brown color fiber (BCF). First, 21 spots were associated with secondary metabolism; 15 of these with high abundance in BCF were involved in flavonoid biosynthesis. Second, several spots with lower abundance in BCF were found. Thirteen spots were related to energy/carbohydrate metabolism; in particular, spots related to the glycolytic pathway exhibited lower abundance in 12 DPA BCF. Several spots related to redox homeostasis, cytoskeleton, and protein metabolism also showed lower abundance in BCF, including proteins that are critical for fiber development, such as ascorbate peroxidase, superoxide dismutase, actin, annexin and heat shock protein. Third, several proteins such as leucine aminopeptidase preprotein and progesterone-5-beta-reductase were newly identified proteins in cotton fibers. These findings demonstrated the presence of a complicated metabolic network in BCF and advanced our understanding of the molecular mechanisms of pigment biosynthesis in colored cotton, which will provide new insight for the development of new color types by genetic manipulation.Highlights► Comparative proteomic analysis revealed the differences between BCF and WCF. ► Protein spots involved in flavonoid biosynthesis showed higher abundance in BCF. ► Differences of several other spots showed complicated metabolic network in BCF. ► Clues were provided for pigment biosynthesis based on proteomics.

Cotton fiber is an ideal model for studying plant cell elongation. To date, the underlying mechanisms controlling fiber elongation remain unclear due to their high complexity. In this study, a comparative proteomic analysis between a short-lint fiber mutant (Ligon lintless, Li1) and its wild-type was performed to identify fiber elongation-related proteins. By 2-DE combined with local EST database-assisted MS/MS analysis, 81 differentially expressed proteins assigned to different functional categories were identified from Li1 fibers, of which 54 were down-regulated and 27 were up-regulated. Several novel aspects regarding cotton fiber elongation can be illustrated from our data. First, over half of the down-regulated proteins were newly identified at the protein level, which is mainly involved in protein folding and stabilization, nucleocytoplasmic transport, signal transduction, and vesicular-mediated transport. Second, a number of cytoskeleton-related proteins showed a remarkable decrease in protein abundance in the Li1 fibers. Accordingly, the architecture of actin cytoskeleton was severely deformed and the microtubule organization was moderately altered, accompanied with dramatic disruption of vesicle trafficking. Third, the expression of several proteins involved in unfolded protein response (UPR) was activated in Li1 fibers, indicating that the deficiency of fiber cell elongation was related to ER stress. Collectively, these findings significantly advanced our understanding of the mechanisms associated with cotton fiber elongation.

The gene GhCPK1 encoding a calcium dependent protein kinase was identified from cotton. Transcripts of GhCPK1 accumulated primarily in the elongating fiber, and Arabidopsis plants transformed with GhCPK1 promoter-GUS construct exhibited GUS activity mainly in the developing trichomes, roots, young leaves and sepals. In the bombarded onion epidermal cells, GhCPK1-GFP fusion proteins showed a subcellular distribution in the plasma membrane. In vitro assays indicated that GhCPK1 was a functional calcium-dependent kinase able to undergo autophosphorylation and phosphorylation of the known substrate histone III-S. Together, these results suggest that GhCPK1 may play a role in the calcium signaling events associated with fiber elongation.

The dehydration-responsive element binding (DREB) transcription factors play central roles in regulating expression of stress-inducible genes under abiotic stresses. In the present work, PpDBF1 (Physcomitrella patensDRE-binding Factor1) containing a conserved AP2/ERF domain was isolated from the moss P. patens. Sequence comparison and phylogenetic analysis revealed that PpDBF1 belongs to the A-5 group of DREB transcription factor subfamily. The transcriptional activation activity and DNA-binding specificity of PpDBF1 were verified by yeast one-hybrid and electrophoretic mobility shift assay experiments, and its nuclear localization was demonstrated by particle biolisitics. PpDBF1 transcripts were accumulated under various abiotic stresses and phytohormones treatments in P. patens, and transgenic tobacco plants over-expressing PpDBF1 gained higher tolerance to salt, drought and cold stresses. These results suggest that PpDBF1 may play a role in P. patens as a DREB transcription factor, implying that similar regulating systems are conserved in moss and higher plants.

The transition from the vegetative phase to the reproductive phase is a major developmental process in flowering plants. The underlying mechanism controlling this cellular process remains a research focus in the field of plant molecular biology. In the present work, we identified a gene encoding the C3H2C3-type RING finger protein NtRCP1 from tobacco BY-2 cells. Enzymatic analysis demonstrated that NtRCP1 is a functional E3 ubiquitin ligase. In tobacco plants, expression level of NtRCP1 was higher in the reproductive shoot apices than in the vegetative ones. NtRCP1-overexpressing plants underwent a more rapid transition from the vegetative to the reproductive phase and flowered markedly earlier than the wild-type control. Histological analysis revealed that the shoot apical meristem of NtRCP1-overexpressing plants initiated inflorescence primordia precociously compared to the wild-type plant due to accelerated cell division. Overexpression of NtRCP1 in BY-2 suspension cells promoted cell division, which was a consequence of the shortened G2 phase in the cell cycle. Together, our data suggest that NtRCP1 may act as a regulator of the phase transition, possibly through its role in cell cycle regulation, during vegetative/reproductive development in tobacco plant.

In this study, we identified a defense-related major latex protein (MLP) from upland cotton (designated GhMLP28) and investigated its functional mechanism. GhMLP28 transcripts were ubiquitously present in cotton plants, with higher accumulation in the root. Expression of the GhMLP28 gene was induced by Verticillium dahliae inoculation and was responsive to defense signaling molecules, including ethylene, jasmonic acid, and salicylic acid. Knockdown of GhMLP28 expression by virus-induced gene silencing resulted in increased susceptibility of cotton plants to V. dahliae infection, while ectopic overexpression of GhMLP28 in tobacco improved the disease tolerance of the transgenic plants. Further analysis revealed that GhMLP28 interacted with cotton ethylene response factor 6 (GhERF6) and facilitated the binding of GhERF6 to GCC-box element. Transient expression assay demonstrated that GhMLP28 enhanced the transcription factor activity of GhERF6, which led to the augmented expression of some GCC-box genes. GhMLP28 proteins were located in both the nucleus and cytoplasm and their nuclear distribution was dependent on the presence of GhERF6. Collectively, these results demonstrate that GhMLP28 acts as a positive regulator of GhERF6, and synergetic actions of the two proteins may contribute substantially to protection against V. dahliae infection in cotton plants.