Characterization of the Gh4CL gene family reveals a role of Gh4CL7 in drought tolerance.

BACKGROUND: The function of 4-coumarate-CoA ligases (4CL) under abiotic stresses has been studied in plants, however, limited is known about the 4CL genes in cotton (G. hirsutum L.) and their roles in response to drought stress. RESULTS: We performed genome-wide identification of the 4CL genes in G. hirsutum and investigated the expression profiles of the identified genes in various cotton tissues and in response to stress conditions with an aim to identify 4CL gene(s) associated with drought tolerance. We identified 34 putative 4CL genes in G. hirsutum that were clustered into three classes. Genes of the same class usually share a similar gene structure and motif composition. Many cis-elements related to stress and phytohormone responses were found in the promoters of the Gh4CL genes. Of the 34 Gh4CL genes, 26 were induced by at least one abiotic stress and 10 (including Gh4CL7) were up-regulated under the polyethylene glycol (PEG) simulated drought stress conditions. Virus-induced gene silencing (VIGS) in cotton and overexpression (OE) in Arabidopsis thaliana were applied to investigate the biological function of Gh4CL7 in drought tolerance. The Gh4CL7-silencing cotton plants showed more sensitive to drought stress, probably due to decreased relative water content (RWC), chlorophyll content and antioxidative enzyme activity, increased stomatal aperture, and the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2). Arabidopsis lines overexpressing Gh4CL7, however, were more tolerant to drought treatment, which was associated with improved antioxidative enzyme activity, reduced accumulation of MDA and H2O2 and up-regulated stress-related genes under the drought stress conditions. In addition, compared to their respective controls, the Gh4CL7-silencing cotton plants and the Gh4CL7-overexpressing Arabidopsis lines had a ~ 20% reduction and a ~ 10% increase in lignin content, respectively. The expression levels of genes related to lignin biosynthesis, including PAL, CCoAOMT, COMT, CCR and CAD, were lower in Gh4CL7-silencing plants than in controls. Taken together, these results demonstrated that Gh4CL7 could positively respond to drought stress and therefore might be a candidate gene for improvement of drought tolerance in cotton. CONCLUSION: We characterized the 4CL gene family in upland cotton and revealed a role of Gh4CL7 in lignin biosynthesis and drought tolerance.

The endochitinase VDECH from Verticillium dahliae inhibits spore germination and activates plant defense responses.

Chitinases function in the digestion of chitin molecules, which are present principally in insects and fungi. In plants, chitinase genes play important roles in defense, and their expression can be triggered in response to both biotic and abiotic stresses. In this study, we cloned and characterized an endochitinase (VDECH) from Verticillium dahliae, strain Vd080. The VDECH coding region consists of 1845bp with two exons and one 54bp intron, encoding a 615 amino acid protein with the predicted molecular weight (MW) of 63.9kDa. The VDECH cDNA without signal peptide-encoding region was introduced into pCold-TF vector and the recombinant protein HIS-VDECH with a predicted MW of ∼114kDa was expressed. HIS-VDECH showed high tolerance to extreme temperature, exhibiting efficient chitinolytic activity at 50°C. In addition, VDECH triggered typical plant defense responses, including a hypersensitive response, oxidative burst, and elicited increased expression of defense-related genes in both Arabidopsis and cotton. VDECH-treatment of the conidial spores of V. dahliae and Fusarium oxysporum resulted in marked reductions in the germination of these spores in both fungi. After 36h of incubation with VDECH, the inhibition rate of germination was recorded at 99.57% for V. dahliae, and 96.89% for F. oxysporum. These results provide evidence that VDECH is recognized by the plant to elicit defense responses, and also that VDECH is an effective inhibitor of conidia germination, both of which may be exploited for disease control.

Functional Analysis of the Pathogenicity-Related Gene VdPR1 in the Vascular Wilt Fungus Verticillium dahliae.

Verticillium dahliae Kleb., the causal agent of vascular wilt, can seriously diminish the yield and quality of many crops, including cotton. The pathogenic mechanism to cotton is complicated and unclear now. To screen pathogencity related genes and identify their function is the reliable way to explain the mechanism. In this study, we obtained a low-pathogenicity mutant vdpr1 from a T-DNA insertional library of the highly virulent isolate of V. dahliae Vd080, isolated from cotton. The tagged gene was named pathogenicity-related gene (VdPR1). The deletion mutant ΔVdPR1 did not form microsclerotia and showed a drastic reduction in spore yield and mycelial growth, compared to wild type. Also, ΔVdPR1 showed significantly lower protease and cellulase activities than those of wild type. Complementation of the mutant strain with VdPR1 (strain ΔVdPR1-C) almost completely rescued the attributes described above to wild-type levels. The knockout mutant ΔVdPR1 showed delayed infection, caused mild disease symptoms, formed a smaller biomass in roots of the host, and showed compromised systemic invasive growth in the xylem. These results suggest that VdPR1 is a multifaceted gene involved in regulating the growth development, early infection and pathogenicity of V. dahliae.

VdCYC8, Encoding CYC8 Glucose Repression Mediator Protein, Is Required for Microsclerotia Formation and Full Virulence in Verticillium dahliae.

Verticillium dahliae is the primary causal agent for Verticillium wilt disease on a diverse array of economically important crops, including cotton. In previous research, we obtained the low-pathogenicity mutant T286 from the T-DNA insertional mutant library of the highly virulent isolate Vd080 derived from cotton. In this study, the target disrupted gene VdCYC8 was identified by TAIL-PCR, encoding a homolog of CYC8 proteins involved in glucose repression. The deletion mutant ΔCYC8 exhibited several developmental deficiencies, including reduced microsclerotia formation, reduced sporulation, and slower growth. Moreover, compared with the wild type strain Vd080, the pathogenicity of strain ΔCYC8 was significantly decreased on cotton seedlings. However, the complementary mutants ΔCYC8-C led to restoration of the wild type phenotype or near wild type levels of virulence on cotton. Interestingly, pathogenicity of the strains was correlated with VdCYC8 gene expression levels in complemented mutants. Gene expression analyses in the wild type strain Vd080, the ΔCYC8-45 strain, and complemented strain ΔCYC8-C26 indicated that VdCYC8 regulates the transcription levels of several genes in V. dahliae that have roles in melanin and production.

[Effects of transgenic cotton expressing chitinase and glucanase genes on the diversity of soil bacterial community].

The transgenic cotton expressing chitinase and glucanase genes was studied using nontransgenic cotton as a control. Specifically, the effects of exogenous genes on bacterial community diversity in rhizospheres of cotton at stages of seedling, budding, boll forming and boll opening were evaluated through comparing the number of cultivable bacteria and analyzing 16S rRNA gene clone libraries. The results showed that the number of cultivable bacteria was not affected by exogenous genes but the cotton growth period, and the number peaked at the stage of boll forming with vigorous metabolism. The 16S rRNA gene clone library prepared from soil bacteria in rhizospheres of transgenic and nontransgenic cotton at different stages contained 2400 clones which covered 283 genera. Among them, Acidobacterium was the most dominant group which contained 642 clones, followed by unclassified bacterium and Flavisolibacter. Compared with nontransgenic cotton, the rhizosphere bacterial diversity of transgenic cotton exhibited lower level at the same growth stage, however, their common bacterial communities increased with growth and development. Our results suggest that chitinase and glucanase genes decrease the rhizosphere bacterial diversity at distinct degrees, however, the difference of bacterial diversity between transgenic and nontransgenic cotton reduces gradually with the extension of cultivation period.

Isolation and functional analysis of the pathogenicity-related gene VdPR3 from Verticillium dahliae on cotton.

The fungal plant pathogen Verticillium dahliae is the causal agent of vascular wilt, a disease that can seriously diminish cotton fiber yield. The pathogenicity mechanism and the identity of the genes that interact with cotton during the infection process still remain unclear. In this study, we investigated the low-pathogenic, non-microsclerotium-producing mutant vdpr3 obtained in a previous study from the screening of a T-DNA insertional library of the highly virulent isolate Vd080; the pathogenicity-related gene (VdPR3) in wild-type strain Vd080 was cloned. Knockout mutants (ΔVdPR3) showed lower mycelium growth and obvious reduction in sporulation ability without microsclerotium formation. An evaluation of carbon utilization in mutants and wild-type isolate Vd080 demonstrated that mutants-lacking VdPR3 exhibited decreased cellulase and amylase activities, which was restored in the complementary mutants (ΔVdPR3-C) to levels similar to those of Vd080. ΔVdPR3 postponed infectious events in cotton and showed a significant reduction in pathogenicity. Reintroduction of a functional VdPR3 copy into ΔVdPR3-C restored the ability to infect cotton plants. These results suggest that VdPR3 is a multifunctional gene involved in growth development, extracellular enzyme activity, and virulence of V. dahliae on cotton.

Expression and significance of hypoxemia-inducible factor-1alpha in osteosarcoma of the jaws.

OBJECTIVE: The objective of this study was to examine the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in osteosarcoma of the jaw (OSJ) and its relationships with tumoral angiogenesis and clinicopathologic characteristics. STUDY DESIGN: Streptavidin peroxidase immunohistochemical staining was used to detect the expression of HIF-1alpha and CD34 in paraffin-embedded samples from 25 patients. There were 14 male and 11 female patients, ages ranged from 3 to 63 years. RESULTS: HIF-1alpha was overexpressed in OSJ. HIF-1alpha expression was correlated with tumoral microvascular density, as well as with the size, pathologic grade, and clinical stage of OSJ. Expression of HIF-1alpha was also correlated with clinicopathologic characteristics, such as primary occurrence or recurrence of the tumor, but not with metastasis. CONCLUSION: HIF-1alpha was significantly correlated with the size, pathologic grade, clinical stage, and primary occurrence or recurrence of the OSJ. HIF-1alpha appeared to promote tumoral angiogenesis in OSJ and may be an important target in antitumoral therapy.