Identification of genes that are preferentially expressed in conidiogenous cell development of Metarhizium anisopliae by suppression subtractive hybridization

Abstract

The insect pathogenic fungus Metarhizium anisopliae is widely used as an insect biocontrol agent. The M. anisopliae conidium plays an important role in pathogenesis and disease transmission. The aim of this study was to identify genes whose expression is up-regulated during conidiogenous cell development. This is a powerful strategy for obtaining insight into the molecular events that regulate conidiation. We isolated genes that are preferentially expressed in the developing conidiophores of the common fungal locust pathogen M. anisopliae CQMa102 using suppression subtractive hybridization. Based on the results of cDNA array dot blotting, we identified 109 unique expressed sequence tags (ESTs) that were up-regulated more than fivefold during conidiophore formation. Among these 109 ESTs were 45 (41.3%) with significant similarity to NCBI annotated hypothetical proteins, 35 (32.1%) with low similarity to known or predicted genes that might represent novel genes, and 29 (26.6%) with significant similarity to known proteins involved in various cell and molecular processes, such as ell structure and function, cell metabolism, protein metabolism, stress response, nucleic acid metabolism, and cell cycle and growth. We confirmed the up-regulation of 11 randomly selected genes with real-time reverse transcriptase-PCR analysis. The results of this study provide a preliminary description of genes that may be involved in the molecular regulation of fungal conidiogenesis.

Genetically altering the expression of neutral trehalase gene affects conidiospore thermotolerance of the entomopathogenic fungus Metarhizium acridum

“…A standard curve was made to optimize the amplification efficiency with the primer pairs L1 (5'-GCACAAGAAGATACCGATGGC-3') and L2 (5'-CGATCCACTGGGTTCTCATTTA-3'). Gdpdh encoding gly ceraldehyde-3-phosphate dehydrogenase was selected as an internal control, and the primers of 5'-AGATGGAGGAGTTGGTGTTG-3' and 5'-GACTGCCCGCATTGAGAAG-3' were used for it [47]. The cycling conditions were 95°C for 3 min followed by 45 cycles of 95°C for 10 sec, annealing at 59°C ( Ntl ) or 60°C ( Gdpdh ) for 10 sec.…”

“…Data were square root arcsine transformed before being subjected to analysis of variance (ANOVA) for a completely randomized design. The means were separated using Tukey's multiple range test, carried out using DPS software [47]. Statistical significance was established at p < 0.05.…”

Secondary metabolites from entomopathogenic Hypocrealean fungi

“…341 Data from subtractive cDNA libraries of M. anisopliae identified genes differentially expressed during growth on hemolymph, 342 during microcycle conidiation, 343 and in conidiogenous cells. 344 Although the number of unigenes reported in these studies is small, some ESTs were identified that putatively encode secondary metabolite biosynthetic enzymes. [341][342][343][344][345] 3.5.2 Transcriptome studies with Beauveria bassiana.…”

“…344 Although the number of unigenes reported in these studies is small, some ESTs were identified that putatively encode secondary metabolite biosynthetic enzymes. [341][342][343][344][345] 3.5.2 Transcriptome studies with Beauveria bassiana. EST sequencing of aerial conidia, submerged conidia and in vitro blastospores of B. bassiana revealed a highly plastic transcriptome with major differences among the different libraries.…”

Enhancing the Biocontrol Potential of the Entomopathogenic Fungus in Multiple Respects via the Overexpression of a Transcription Factor Gene MaSom1

“…The mature conidia from 1/4 SDAY plates were washed with 1% PBS and harvested to detect the expression levels of MaSom1 in WT, OE-4, OE-34, OE-55 and VT by qRT-PCR with the primer pair of MaSom1-QF/MaSom1-QR (Table S1). Total RNA extraction, cDNA reverse transcription, and qRT-PCR were performed according to the existing method [55]. The glyceraldehyde 3-phosphate dehydrogenase gene gpdh (GenBank accession No.…”