| Metarhizium anisopliae is a significant filamentous pathogenic fungus, because it infects ca. 200 insect species from more than 50 taxonomic families, including termites, grasshoppers, thrips and mosquitoes. The mechanisms by which the insect pathogenic fungus M. anisopliae infects the host insect are relatively well understood. Conidiation, as the efficient propagule used in biocontrol programs, is a key step for survival and transmission in the disease cycle. Just like two representative conidiation elucidated in detail for two model organisms: Neuropora crassa and Aspergillus nidulans, our previous work found that M. anisopliae also exhibited two different conidiation programs: normal conidiation on non-inducing medium and microcycle conidiation on inducing medium. Compared with normal conidiation, microcycle conidiation showed remarkable advantages in the speed of conidia production and resistance to stress factors. Therefore, better understanding of the molecular mechanism of microcycle conidiation would improve conidial commercial production of M. anisopliae, which would greatly facilitate expansion of the scope of insect fungal pathogen applications in pest control.Previously, genes preferentially expressed during microcycle conidiation vs. in normal conidiation hyphae were isolated by suppression subtractive hybridization in M. anisopliae. Among these candidates, we identified and described three key genes (AGA, Pyk, PP5), which are highly expressed during microcycle conidiation. The results provide the ideal candidates for further studies of microcycle conidiation and its molecular regulation. The main achievements are as follows:(一)MaAGA regulates microcycle conidiation and thermotolerance in M. anisopliae1.A full-length arginase gene MaAGA was cloned and sequenced, and deposited in Genbank under accession number (HM068369). The ORF (open reading frame), which was interrupted by two introns, was 978 bp long. Analysis of the predicted MaAGA protein indicated that it was composed of 325 amino acid residues (35.05 kDa) with a predicted pI of 5.10. The predicted protein encoded by MaAGA contained two manganese ion binging domain, arginine-specific binding domain and other arginase-conserved amino acid residues. Identities with the MaAGA sequence were 21–23% (plant) and 30–38% (bacterium) compared to 40–53% and 40–49% for fungal and animal arginases, respectively.2.A gene interference strategy was used and the RNAi vector containing a 367 bp fragment of MaAGA was transformed into Metarhizium wild type. Five putative RNAi-MaAGA mutants were identified. To investigate that RNA interference efficiency of MaAGA in mutants, we verified mRNA expression and arginase activity of MaAGA in mutants. These results demonstrated a successful suppression of MaAGA in mutants (22%-62%)3.On SYA medium, the conidiation processes of RNAi mutant obviously differed from the wild type and exhibited an unexpected phenotype, in which daughter cells had partly failed to separate from their mother cells. After further rounds of budding, the mutant formed tree-like structure. The growth of the RNAi mutant-3 (2.01±0.10 cm) was significantly slower than that of the wild type (2.56±0.9 cm) (P < 0.05). Besides, more similar white hyphae could be observed after 5 day postinoculation on SYA medium. In the phenotype restore assay, when the medium was amended with ornithine, the fungal phenotype could be restored, which supports a direct role for arginase in microconidial formation. Further, we investigated nine daughter-special genes expression in RNAi mutant. Mob2 gene, a pivotal protein involved cell separation system, was down-regulated >2-fold in the RNAi mutant. However, normal conidiation seemed to be unaffected in RNAi mutant compared with the wild type.4.We investigate the difference of conidial production. Both the total conidial yield (at day 11, P= 0.008) and the speed of conidiation (at day 2, P= 0.005) /cm~2 of the RNAi mutant-3 were significantly lower. The mean total conidial yield and speed of the RNAi mutant-3 were 0.27-fold and 0.32-fold higher than those of wild-type, respectively. On normal conidiation medium, the total conidial yield (at day 12, P = 0.593) and the speed of conidiation (at day 2, P= 0.674) in RNAi mutant-3 and wild-type were not discernibly different.5.The conidial thermotolerances were analyzed with dry-heat, wet-heat and UV-B treatment. The germination of RNAi mutant was significantly lower than that of wild type under dry-heat, wet-heat treatment, except UV-B. Trehalose was an important component in fungal conidia and had been widely reported to protect the cells against several stresses. We investigated conidial accumulation of trehalose and found trehalose content was about 3.31 pg/conidia in RNAi mutant and 3.86 pg/conidia in wild type, showing about 14.3% reduction compared with the wild type Furthermore, RT-PCR showed the decreasing expression of ntl (Neutral trehalase) resulted in lower trehalose content in RNAi mutants.6 . No significant difference was found in appressorium formation and pathogenicity between the wild-type and RNAi mutant-3, indicating that MaAGA was dispensable to conidial tolerance to UV-B, appressorium formation and pathogenicity.(二)Pyk regulates conidial shape of microcycle conidiation in M. anisopliae1.A full-length arginase gene Pyk and its upstream and downstream regulatory sequences was cloned and sequenced, and deposited in Genbank under accession number (HM068369). The ORF with no interruption was 978 bp long. Analysis of the predicted Pyk protein indicated that it was composed of 583 amino acid residues (47.8 kDa) with a predicted pI of 9.73. No signal peptide was found in the prediceted protein. The phylogenetic tree showed 63%-77% identies between Pyk and the selected 17 species.2.The optimal RNA interference area of Pyk was designed and inserted into the RNAi vector, and then the expression vector was transformed into Metarhizium wild type. Three putative RNAi-Pyk mutants were identified. To investigate that RNA interference efficiency of Pyk in mutants, we verified mRNA expression of Pyk in mutants. These results demonstrated a successful suppression of Pyk in mutants (33%-56%)3.Furthermore, we analyzed suppression effect to Pyk in mutations. On SYA, The wild type has only Ellipsoid conidia. But the mutants showed more varied shapes of conidia: Ellipsoid conidia and Rod-like conidia. Moreover less white hypha in colonial morphology could be observed compared to wild type.(三)Cloning and expression analysis of Ser/Thr protein phosphatase type 5 during microcycle conidiation in M. ansopliae1.A full-length cDNA and DNA of PP5 was cloned and sequenced, and deposited in Genbank under accession number (HM068369). The genomic DNA was 2100bp. The ORF of cDNA with six interruptions was 1428 bp long.2.The predicted PP5 protein was composed of 475 amino acid residues (38.1 kDa) with a predicted pI of 9.66. No signal peptide was found in the prediceted protein. In protein structure analysis, Ser/Thr protein phosphatase type 5 in M. anisopliae showed a conserved structure features.3.Quantitative real time PCR analysis showed that PP5 expression varied obviously in different stages of microcycle conidiation. Expression was sharply up-regulated after 16 h, with the highest transcript levels at 24 h in microcycle conidiation, but lowly expressed in normal conidiation. |
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