Proteomic and virus-induced gene silencing (VIGS) Analyses reveal that gossypol, brassinosteroids, and jasmonic acid contribute to the resistance of cotton to Verticillium dahliae.

Verticillium wilt causes massive annual losses of cotton yield, but the mechanism of cotton resistance to Verticillium dahliae is complex and poorly understood. In this study, a comparative proteomic analysis was performed in resistant cotton (Gossypium barbadense cv7124) on infection with V. dahliae. A total of 188 differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) analysis and could be classified into 17 biological processes based on Gene Ontology annotation. Most of these proteins were implicated in stimulus response, cellular processes and metabolic processes. Based on the proteomic analysis, several genes involved in secondary metabolism, reactive oxygen burst and phytohormone signaling pathways were identified for further physiological and molecular analysis. The roles of the corresponding genes were further characterized by employing virus-induced gene silencing (VIGS). Based on the results, we suggest that the production of gossypol is sufficient to affect the cotton resistance to V. dahliae. Silencing of GbCAD1, a key enzyme involving in gossypol biosynthesis, compromised cotton resistance to V. dahliae. Reactive oxygen species and salicylic acid signaling may be also implicated as regulators in cotton responsive to V. dahliae according to the analysis of GbSSI2, an important regulator in the crosstalk between salicylic acid and jasmonic acid signal pathways. Moreover, brassinosteroids and jasmonic acid signaling may play essential roles in the cotton disease resistance to V. dahliae. The brassinosteroids signaling was activated in cotton on inoculation with V. dahliae and the disease resistance of cotton was enhanced after exogenous application of brassinolide. Meanwhile, jasmonic acid signaling was also activated in cotton after inoculation with V. dahliae and brassinolide application. These data provide highlights in the molecular basis of cotton resistance to V. dahliae.

Genes expression analyses of sea-island cotton (Gossypium barbadense L.) during fiber development.

Sea-island cotton (Gossypium barbadense L.) is one of the most valuable cotton species due to its silkiness, luster, long staples, and high strength, but its fiber development mechanism has not been surveyed comprehensively. We constructed a normalized fiber cDNA library (from -2 to 25 dpa) of G. barbadense cv. Pima 3-79 (the genetic standard line) by saturation hybridization with genomic DNA. We screened Pima 3-79 fiber RNA from five developmental stages using a cDNA array including 9,126 plasmids randomly selected from the library, and we selected and sequenced 929 clones that had different signal intensities between any two stages. The 887 high-quality expressed sequence tags obtained were assembled into 645 consensus sequences (582 singletons and 63 contigs), of which 455 were assigned to functional categories using gene ontology. Almost 50% of binned genes belonged to metabolism functional categories. Based on subarray analysis of the 887 high-quality expressed sequence tags with 0-, 5-, 10-, 15-, and 20-dpa RNA of Pima 3-79 fibers and a mixture of RNA of nonfiber tissues, seven types of expression profiles were elucidated. Furthermore our results showed that phytohormones may play an important role in the fiber development.

[Spontaneous and induced programmed cell death in suspension cell cultures of cotton (Gossypium hirsutum L.)].

Cotton suspension cells grew well in the MS medium supplemented with 0.1 mg/L 2,4 D and 0.1 mg/L KT. Senescence occurred when the cells were unsubcultured. The cells began to lose their viabilities on the 17th day, and on the 21th day oligonucleosomal sized DNA fragments ( DNA ladder) could be detected. Oligonucleosomal sized DNA fragments ( DNA ladder) was the hallmark of the programmed cell death. Programmed cell death of cotton suspension cells could be induced respectively by some stress factors which included heatshock (42+/-3 degrees C for 8 hours), 10 micromol/L camptothecin, 20 micromol/L fumonisin B1 and 50 mmol/L cycloheximide. The cotton suspension cells which grew in the MS medium supplemented with 0.1 mg/L 2,4 D and 0.1 mg/L KT differred physiologically from the cells in the MS medium supplemented with 0.1 mg/L IBA and 0.1 mg/L KT, and they responded differentially to the heatshock, 10 micromol/L camptothecin and 20 micromol/L fumonisin B1, while the same to 50 mmol/L cycloheximide.

[Construction and analysis of SSH library of Gossypium barbadense upon infection with Verticillium dahliae].

Roots were collected from the seedlings inoculated with pathogen Verticillium dahliae after 2, 4, 8, 12, 24, 48, 72 and 96 hours for total RNA extraction. The cDNAs from the inoculated seedlings were used as the tester and those from the control seedlings as the driver. SSH method was employed to find the differently expressed cDNAs responding to the pathogen. T/A clone library was constructed containing 534 clones. The cDNA inserts were amplified from the bacterial clones directly with M13 primers by PCR. The size of the products ranged 0.2 - 1.2 kb with an average size of 0.5 kb. The SSH products were dotted on nylon filters, and the positive clones were screened by virtual Northern blotting with probes of the two kinds of initiative cDNAs. Totally 78 clones which were up-regulated and putatively involved in the defense response of G. barbadense were identified and sequenced. Sequence similarity searches were performed with the Blastn and Blastx. Most of them showed high or partial homology to genes or ESTs induced by different stresses in Arabidopsis thaliana and other species,such as the pathogenesis-related 10 family of G. hirsumtum and disease resistance-responsive family protein in Arabidopsis thaliana. The results would be helpful to understand the molecular mechanisms of disease response in cotton.