Generation of transcriptome profiling and gene functional analysis in Gossypium hirsutum upon Verticillium dahliae infection.

Verticillium wilt caused by the soil-borne fungus Verticillium dahliae, is a devastating disease in cotton. To get more candidate genes related to wilt resistance, a normalized, full-length cDNA library was used to generate the transcriptome profile from a wilt-resistant Gossypium hirsutum variety (Zhongzhimian KV3) upon V. dahliae strain V991 infection. Total 3486 high-quality ESTs were focused from randomly selected 4000 clones, which included 3065 singletons and 421 contigs. To categorize these unigenes, they were compared to functional domain databases. Total 106 unigenes were found to be homologous to known defense-related genes. Among them, PR genes were the major group. Furthermore, knockdown of GhWRKY22, GhWRKY33, GhChitinase, GhCML, and GhDirigent resulted in increased susceptibility of resistant cotton to V. dahliae infection. The results of transcriptome profiles and virus induced gene silencing (VIGS) analysis laid a good foundation for further investigation of cotton resistance to wilt disease.

Cotton gene expression profiles in resistant Gossypium hirsutum cv. Zhongzhimian KV1 responding to Verticillium dahliae strain V991 infection.

Verticillium wilt of cotton (Gossypium hirsutum) is a widespread and destructive disease that is caused by the soil-borne fungus pathogen Verticillium dahliae (V. dahliae). To study the molecular mechanism in wilt tolerance, suppression subtractive hybridization (SSH) and dot blot techniques were used to identify the specifically expressed genes in a superior wilt-resistant cotton cultivar (G. hirsutum cv. Zhongzhimian KV1) after inoculation with pathogen. cDNAs from the root tissues of Zhongzhimian KV1 inoculated with V. dahliae strain V991 or water mock were used to construct the libraries that contain 4800 clones. Based on the results from dot blot analysis, 147 clones were clearly induced by V. dahliae and selected from the SSH libraries for sequencing. A total of 92 up-regulated and 7 down-regulated non-redundant expressed sequences tags (ESTs) were identified as disease responsive genes and classified into 9 functional groups. Two important clues regarding wilt-resistant G. hirsutum were obtained from this study. One was Bet v 1 family; the other was UbI gene family that may play an important role in the defense reaction against Verticillium wilt. The result from real-time quantitative reverse transcription polymerase chain reaction showed that these genes were activated quickly and transiently after inoculation with V. dahliae.

Dominant gene cpls(r)1 corresponding to premature leaf senescence resistance in cotton (Gossypium hirsutum L.).

Cotton (Gossypium hirsutum L.) premature leaf senescence-resistant inbred XLZ33 and senescence-susceptible inbred lines XLZ13 were selected and crossed to produce F(1), F(1)-reciprocal, F(2) and BC(1) generations for evaluation of leaf senescence process and inheritance. The results showed that leaf senescence processes for XLZ13 and XLZ33 were obviously different and leaf senescence traits could be distinguished between the two parents at particular periods of cotton growth. Inheritance anlysis for the cotton premature leaf senescence resistant trait further showed that the segregation in the F(2) fit a 3:1 ratio inheritance pattern, with resistance being dominant. The backcross of F(1) to the susceptible parent produced a 1:1 ratio, confirming that cotton premature leaf senescence resistant trait was from a single gene. The single dominant gene controlling cotton premature leaf senescence resistance in XLZ33 was named as cotton premature leaf senescence resistance 1, with the symbol cpls(r)1.

Proteomic analysis of the sea-island cotton roots infected by wilt pathogen Verticillium dahliae.

Verticillium wilt of cotton is a vascular disease mainly caused by the soil-born filamentous fungus Verticillium dahliae. To study the mechanisms associated with defense responses in wilt-resistant sea-island cotton (Gossypium barbadense) upon V. dahliae infection, a comparative proteomic analysis between infected and mock-inoculated roots of G. barbadense var. Hai 7124 (a cultivar showing resistance against V. dahliae) was performed by 2-DE combined with local EST database-assisted PMF and MS/MS analysis. A total of 51 upregulated and 17 downregulated proteins were identified, and these proteins are mainly involved in defense and stress responses, primary and secondary metabolisms, lipid transport, and cytoskeleton organization. Three novel clues regarding wilt resistance of G. barbadense are gained from this study. First, ethylene signaling was significantly activated in the cotton roots attacked by V. dahliae as shown by the elevated expression of ethylene biosynthesis and signaling components. Second, the Bet v 1 family proteins may play an important role in the defense reaction against Verticillium wilt. Third, wilt resistance may implicate the redirection of carbohydrate flux from glycolysis to pentose phosphate pathway (PPP). To our knowledge, this study is the first root proteomic analysis on cotton wilt resistance and provides important insights for establishing strategies to control this disease.

Subtle regulation of cotton resistance to Verticillium wilt mediated by MAPKK family members.

Verticillium wilt caused by soil-borne fungus of Verticillium dahliae Kleb. is one of the most devastating diseases of cotton. Since the hierarchically organized mitogen-activated protein kinase (MAPK) cascade plays pivotal roles in signaling plant defense against pathogen attack, and the key nodes of MAPKKs (MKKs) may serve as for the convergence and divergence of signals in MAPK cascades, the possible relations between MAPK signaling and cotton Verticillium resistance were examined in this study. A total of 24 MKK genes were identified in the Gossypium hirsutum L. genome and then classified based on phylogenetic analysis. Then the regulation roles of all types of cotton MKKs in activation of cotton disease resistance were tested with the virus-induced gene silencing (VIGS) method. The results showed that three types of MKKs (GhMKK4, GhMKK6 and GhMKK9) positively regulate, while GhMKK10 negatively regulate the cotton resistance to Verticillium wilt. Further, more subtle regulation of cotton resistance mediated by MKK genes were revealed. In GhMKK9, only Gh_A12G2448 and Gh_D12G2574 displayed positive regulation of cotton resistance; whereas only Gh_A12G1883 and Gh_D12G2062 displayed negative regulation of cotton resistance in GhMKK10. All these results show that MKK members in MAPK signal cascades play dual roles in subtly regulating of cotton resistance to Verticillium wilt.

Large-scale identification of Gossypium hirsutum genes associated with Verticillium dahliae by comparative transcriptomic and reverse genetics analysis.

Verticillium wilt is a devastating disease of cotton, which is caused by the soil-borne fungus Verticillium dahliae (V. dahliae). Although previous studies have identified some genes or biological processes involved in the interaction between cotton and V. dahliae, its underlying molecular mechanism remains unclear, especially in G. hirsutum. In the present study, we obtained an overview of transcriptome characteristics of resistant upland cotton (G. hirsutum) after V. dahliae infection at 24 h post-inoculation (hpi) via a high-throughput RNA-sequencing technique. A total of 4,794 differentially expressed genes (DEGs) were identified, including 820 up-regulated genes and 3,974 down-regulated genes. The enrichment analysis showed that several important processes were induced upon V. dahliae infection, such as plant hormone signal transduction, plant-pathogen interaction, phenylpropanoid-related and ubiquitin-mediated signals. Moreover, we investigated some key regulatory gene families involved in the defense response, such as receptor-like protein kinases (RLKs), WRKY transcription factors and cytochrome P450 (CYPs), via virus-induced gene silencing (VIGS). GhSKIP35, a partner of SKP1 protein, was involved in ubiquitin-mediated signal. Over-expression of GhSKIP35 in Arabidopsis improved its tolerance to Verticillium wilt in transgenic plants. Collectively, global transcriptome analysis and functional gene characterization provided significant insights into the molecular mechanisms of G. hirsutum-V. dahliae interaction and offered a number of candidate genes as potential sources for breeding wilt-tolerance in cotton.

Selection and heritability of resistance to Bacillus thuringiensis subsp kurstaki and transgenic cotton in Helicoverpa armigera (Lepidoptera: Noctuidae).

Compared with an unselected susceptible population, a cotton bollworm, Helicoverpa armigera (Hübner), population selected for 22 generations with transgenic cotton leaves (modified Cry1A) in the laboratory developed 11.0-fold resistance to Cry1Ac (one single-protein product MVPII). Resistance to Bacillus thuringiensis Berliner subsp kurstaki (Btk) was selected for 22 generations with a 5.2-fold increase in LC50. The estimated realized heritabilities (h2) of resistance for transgenic-cotton- and Btk-selected populations were 0.1008 and 0.2341, respectively. This reflects the higher phenotypic variation in response to Cry1Ac in the transgenic-cotton-selected population. This variation may have been caused by differences in protein toxin levels expressed in different growth stages of the transgenic cotton. Because of the different slopes of the probit regression lines between Cry1Ac and Btk, the estimated realized h2 cannot be used visually to compare resistance development to Cry1Ac and Btk in H armigera. Thus, the response quotient (Q) of resistance was also estimated. The Q values of resistance for transgenic-cotton- and Btk-selected populations were 0.0763 and 0.0836, respectively. This showed that the rate of resistance development would be similar in both selection populations. This result indicates that the selection of resistance using transgenic cotton is different from that selected using the single toxin. Resistance risk to transgenic cotton and Btk in field populations was assessed assuming different pressures of selection by using the estimated h2. Assuming the h2 of resistance in a field population was half of the estimated h2, and the population received prolonged and uniform exposure to transgenic cotton or Btk causing >70% mortality in each generation, we predicted that resistance would increase 10-fold after <23 generations for Cry1Ac in transgenic cotton-selected-populations and after <21 generations for Btk in Btk-selected populations. Cross-resistance would be expected after <48 generations for Btk in transgenic-cotton-selected populations and after <21 generations for Cry1Ac in Btk-selected population. The results show that the potential to evolve resistance is similar in both transgenic-cotton- and Btk-selected populations, but that cross-resistance development to Btk is slower in transgenic-cotton-selected populations than cross-resistance development to Cry1Ac in Btk-selected populations.

Non-uniform distribution pattern for differentially expressed genes of transgenic rice Huahui 1 at different developmental stages and environments.

DNA microarray analysis is an effective method to detect unintended effects by detecting differentially expressed genes (DEG) in safety assessment of genetically modified (GM) crops. With the aim to reveal the distribution of DEG of GM crops under different conditions, we performed DNA microarray analysis using transgenic rice Huahui 1 (HH1) and its non-transgenic parent Minghui 63 (MH63) at different developmental stages and environmental conditions. Considerable DEG were selected in each group of HH1 under different conditions. For each group of HH1, the number of DEG was different; however, considerable common DEG were shared between different groups of HH1. These findings suggested that both DEG and common DEG were adequate for investigation of unintended effects. Furthermore, a number of significantly changed pathways were found in all groups of HH1, indicating genetic modification caused everlasting changes to plants. To our knowledge, our study for the first time provided the non-uniformly distributed pattern for DEG of GM crops at different developmental stages and environments. Our result also suggested that DEG selected in GM plants at specific developmental stage and environment could act as useful clues for further evaluation of unintended effects of GM plants.

Chilling stress--the key predisposing factor for causing Alternaria alternata infection and leading to cotton (Gossypium hirsutum L.) leaf senescence.

Leaf senescence plays a vital role in nutrient recycling and overall capacity to assimilate carbon dioxide. Cotton premature leaf senescence, often accompanied with unexpected short-term low temperature, has been occurring with an increasing frequency in many cotton-growing areas and causes serious reduction in yield and quality of cotton. The key factors for causing and promoting cotton premature leaf senescence are still unclear. In this case, the relationship between the pre-chilling stress and Alternaria alternata infection for causing cotton leaf senescence was investigated under precisely controlled laboratory conditions with four to five leaves stage cotton plants. The results showed short-term chilling stress could cause a certain degree of physiological impairment to cotton leaves, which could be recovered to normal levels in 2-4 days when the chilling stresses were removed. When these chilling stress injured leaves were further inoculated with A. alternata, the pronounced appearance and development of leaf spot disease, and eventually the pronounced symptoms of leaf senescence, occurred on these cotton leaves. The onset of cotton leaf senescence at this condition was also reflected in various physiological indexes such as irreversible increase in malondialdehyde (MDA) content and electrolyte leakage, irreversible decrease in soluble protein content and chlorophyll content, and irreversible damage in leaves' photosynthesis ability. The presented results demonstrated that chilling stress acted as the key predisposing factor for causing A. alternata infection and leading to cotton leaf senescence. It could be expected that the understanding of the key factors causing and promoting cotton leaf senescence would be helpful for taking appropriate management steps to prevent cotton premature leaf senescence.

Differentially expressed genes distributed over chromosomes and implicated in certain biological processes for site insertion genetically modified rice Kemingdao.

Release of genetically modified (GM) plants has sparked off intensive debates worldwide partly because of concerns about potential adverse unintended effects of GM plants to the agro system and the safety of foods. In this study, with the aim of revealing the molecular basis for unintended effects of a single site insertion GM Kemingdao (KMD) rice transformed with a synthetic cry1Ab gene, and bridging unintended effects of KMD rice through clues of differentially expressed genes, comparative transcriptome analyses were performed for GM KMD rice and its parent rice of Xiushui11 (XS11). The results showed that 680 differentially expressed transcripts were identified from 30-day old seedlings of GM KMD rice. The absolute majority of these changed expression transcripts dispersed and located over all rice chromosomes, and existed physical distance on chromosome from the insertion site, while only two transcripts were found to be differentially expressed within the 21 genes located within 100 kb up and down-stream of the insertion site. Pathway and biology function analyses further revealed that differentially expressed transcripts of KMD rice were involved in certain biological processes, and mainly implicated in two types of pathways. One type was pathways implicated in plant stress/defense responses, which were considerably in coordination with the reported unintended effects of KMD rice, which were more susceptible to rice diseases compared to its parent rice XS11; the other type was pathways associated with amino acids metabolism. With this clue, new unintended effects for changes in amino acids synthesis of KMD rice leaves were successfully revealed. Such that an actual case was firstly provided for identification of unintended effects in GM plants by comparative transciptome analysis.

Ectopic expression of the cotton non-symbiotic hemoglobin gene GhHbd1 triggers defense responses and increases disease tolerance in Arabidopsis.

Plant non-symbiotic hemoglobins (nsHbs) play important roles in a variety of cellular processes. Previous evidence from this laboratory indicates that the expression of a class 1 nsHb gene (GhHb1) from cotton is induced in cotton roots challenged with the Verticillium wilt fungus. The present study examined further the expression patterns of the GhHb1 gene in cotton plants and characterized its in vivo function through ectopic overexpression of the gene in Arabidopsis thaliana. Expression of GhHb1 in cotton plants was induced by exogenously applied salicylic acid, methyl jasmonic acid, ethylene, hydrogen peroxide (H(2)O(2)) and nitric oxide (NO). Ectopic overproduction of GhHb1 in Arabidopsis led to constitutive expression of the defense genes PR-1 and PDF1.2, and conferred enhanced disease resistance to Pseudomonas syringae and tolerance to V. dahliae. GhHb1-transgenic Arabidopsis seedlings were more tolerant to exogenous NO and contained lower levels of cellular NO than the wild-type control. Moreover, transgenic plants with relatively high levels of expression of the GhHb1 gene developed spontaneous hypersensitive lesions on the leaves in the absence of pathogen inoculation. Our results indicate that GhHb1 proteins play a role in the defense responses against pathogen invasions, possibly by modulating the NO level and the ratio of H(2)O(2)/NO in the defense process.