The Zinc Finger Transcription Factor BbCmr1 Regulates Conidium Maturation in Beauveria bassiana.

The entomopathogenic fungus Beauveria bassiana is a typical filamentous fungus and has been used for pest biocontrol. Conidia are the main active agents of fungal pesticides; however, we know little about conidial developmental mechanisms and less about maturation mechanisms. We found that a Zn2Cys6 transcription factor of B. bassiana (named BbCmr1) was mainly expressed in late-stage conidia and was involved in conidium maturation regulation. Deletion of Bbcmr1 impaired the conidial cell wall and resulted in a lower conidial germination rate under UV (UV), heat shock, H2O2, Congo red (CR) and SDS stresses compared to the wild type. Transcription levels of the genes associated with conidial wall components and trehalose synthase were significantly reduced in the ΔBbcmr1 mutant. Further analysis found that BbCmr1 functions by upregulating BbWetA, a well-known transcription factor in the central development of BrlA-AbaA-WetA. The expression of Bbcmr1 was positively regulated by BbBrlA. These results indicated that BbCmr1 played important roles in conidium maturation by interacting with the central development pathway, which provided insight into the conidial development networks in B. bassiana. IMPORTANCE Conidium maturation is a pivotal event in conidial development and affects fungal survival ability under various biotic/abiotic stresses. Although many transcription factors have been reported to regulate conidial development, we know little about the molecular mechanism of conidium maturation. Here, we demonstrated that the transcription factor BbCmr1 of B. bassiana was involved in conidium maturation, regulating cell wall structure, the expression of cell wall-related proteins, and trehalose synthesis. BbCmr1 orchestrated conidium maturation by interplaying with the central development pathway BrlA-AbaA-WetA. BbBrlA positively regulated the expression of Bbcmr1, and the latter positively regulated BbwetA expression, which forms a regulatory network mediating conidial development. This finding was critical to understand the molecular regulatory networks of conidial development in B. bassiana and provided avenues to engineer insect fungal pathogens with high-quality conidia.

Gibberellin overproduction promotes sucrose synthase expression and secondary cell wall deposition in cotton fibers.

Bioactive gibberellins (GAs) comprise an important class of natural plant growth regulators and play essential roles in cotton fiber development. To date, the molecular base of GAs' functions in fiber development is largely unclear. To address this question, the endogenous bioactive GA levels in cotton developing fibers were elevated by specifically up-regulating GA 20-oxidase and suppressing GA 2-oxidase via transgenic methods. Higher GA levels in transgenic cotton fibers significantly increased micronaire values, 1000-fiber weight, cell wall thickness and cellulose contents of mature fibers. Quantitative RT-PCR and biochemical analysis revealed that the transcription of sucrose synthase gene GhSusA1 and sucrose synthase activities were significantly enhanced in GA overproducing transgenic fibers, compared to the wild-type cotton. In addition, exogenous application of bioactive GA could promote GhSusA1 expression in cultured fibers, as well as in cotton hypocotyls. Our results suggested that bioactive GAs promoted secondary cell wall deposition in cotton fibers by enhancing sucrose synthase expression.

Gibberellin 20-oxidase promotes initiation and elongation of cotton fibers by regulating gibberellin synthesis.

Cotton is the leading natural fiber, and gibberellin (GA) is a phytohormone involved in the development of cotton fibers. However, it is largely unknown how the GA content in ovules and fibers is regulated and how the endogenous GA concentration affects fiber development. To address these questions, three GA 20-oxidase homologous genes (GhGA20ox1-3) were cloned and the endogenous bioactive GA content in developing ovules and fibers determined by liquid chromatography-electrospray ionization-mass spectrometry. Real-time reverse transcription PCR (RT-PCR) revealed that GhGA20ox1 expressed preferentially in elongating fibers and that the expression level varied with the endogenous GA content consistently, while GhGA20ox2 and GhGA20ox3 transcripts accumulated mainly in ovules. The GA accumulation kinetics as well as the GhGA20ox expression differed in ovules and the attached fibers, suggesting relatively independent GA regulation system in these two sites. Transgenic cotton, over-expressing GhGA20ox1, showed GA over-production phenotypes with increased endogenous GA levels (especially GA(4)) in fibers and ovules. It also produced significantly more fiber initials per ovule, and fiber lengths was increased compared with the control, which demonstrates that up-regulation of the GhGA20ox1 gene promoted fiber initiation and elongation. Our results suggest that GA 20-oxidase is involved in fiber development by regulating GA levels, and corresponding genes might be employed as target genes for the manipulation of fiber initiation and elongation in cotton.

Cotton flavonoid structural genes related to the pigmentation in brown fibers.

Five flavonoid structural genes, encoding chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase, and anthocyanidin reductase, were cloned from a brown-fiber cotton line (T586). The predicted proteins of these genes exhibit high sequence similarity with corresponding enzymes from various plants. RT-PCR analysis showed these genes are developmentally co-regulated and preferentially expressed in developing fibers of T586. Expression analyses and dimethylaminocinnaldehyde staining demonstrated that high transcript levels of these genes in developing fibers and presence of proanthocyanidins in mature fibers co-segregated with brown fiber in a recombination inbred line population. Our results indicated that the cloned flavonoid structural genes and proanthocyanidins were involved in the pigmentation in brown cotton fibers.

Cloning and characterization of a balsam pear class I chitinase gene (Mcchit1) and its ectopic expression enhances fungal resistance in transgenic plants.

A balsam pear (Momordica charantia L.) chitinase (Mcchit1) was purified and sequenced at the N-terminal. The genomic and cDNA coding sequences of Mcchit1 were cloned by rapid amplification of 3' cDNA ends (3'-RACE) and the Y-shaped adaptor dependent extension (YADE) method. Sequence analysis showed that the Mcchit1 protein is a class I chitinase containing a chitin-binding domain and a catalytic domain, but no C-terminal extension. Northern blot indicated that the Mcchit1 transcription is wound-inducible. Overexpression of Mcchit1 dramatically increased intercellular and intracellular endochitinase activities, suggesting that the Mcchit1 gene encodes a secretory endochitinase. It was also found that overexpression of Mcchit1 significantly enhanced resistance to the plant pathogenic fungus Phytophthora nicotianae in transgenic N. benthamiana plants and against Verticillium wilt in transgenic cottons, indicating that the Mcchit1 gene can be a useful gene in plant engineering against fungal diseases.

Functional expression of the cotton gibberellic acid oxidase homologous gene GhGA20ox1 in tobacco.

To determine the physiological function of GhGA20ox1, a homologous gene of GA 20-oxidase from elongating cotton fibers, we expressed this gene ectopically in Nicotiana benthamiana. Reverse transcription-PCR analysis showed that the GhGA20ox1 gene was expressed in the transgenic plants at various levels. It was demonstrated that overexpression of GhGA20ox1 enhanced preferentially the GA(4+7) biosynthesis in N. benthamiana and conferred GA-overproduction characters to transformants. The extent of phenotypic alteration in the transgenic plants was found to correlate with the transcriptional levels of GhGA20ox1 and GA contents. Results indicated that the GhGA20ox1 gene promoted the biosynthesis of the active GAs (GA(4+7)) in transgenic tobacco plants therefore represents a useful gene for manipulating GA levels.

[The cDNA-AFLP differential display in developing anthers between cotton male sterile and fertile line of "Dong A"].

cDNA-AFLP, an effective method for mRNA differential display, was employed to compare the gene expression in developing anthers between the male sterile and fertile plants of cotton (Gossypium hirsutum L.) cultivar, Dong A. In the micro-spore stage, there were more differential bands of cDNA-AFLP than that in the meio-phase stage. Among 64 differential fragments produced by cDNA-AFLP, three were randomly selected for further analysis. RNA dot blotting showed that the GHA27 transcript was expressed mainly in floral tissues; on the other hand, the GHA28 and GHA47 transcripts were present specifically in anther. BLAST analysis demonstrated that GHA27 was highly similar to the plant ADP-ribosylation factor genes, while GHA28 and GHA27 were shown no significant similar to any sequences in the available databases.