Comparative transcriptomic analysis of the heat stress response in the filamentous fungus Metarhizium anisopliae using RNA-Seq

The entomopathogenic fungus Metarhizium anisopliae is widely used for biological control of a variety of insect pests. The effectiveness of the microbial pest control agent, however, is limited by poor thermotolerance. The molecular mechanism underlying the response to heat stress in the conidia of entomopathogenic fungi remains unclear. Here, we conducted high-throughput RNA-Seq to analyze the differential gene expression between control and heat treated conidia of M. anisopliae at the transcriptome level. RNA-Seq analysis generated 6,284,262 and 5,826,934 clean reads in the control and heat treated groups, respectively. A total of 2,722 up-regulated and 788 down-regulated genes, with a cutoff of twofold change, were identified by expression analysis. Among these differentially expressed genes, many were related to metabolic processes, biological regulation, cellular processes and response to stimuli. The majority of genes involved in endocytic pathways, proteosome pathways and regulation of autophagy were up-regulated, while most genes involved in the ribosome pathway were down-regulated. These results suggest that these differentially expressed genes may be involved in the heat stress response in conidia. As expected, significant changes in expression levels of genes encoding heat shock proteins and proteins involved in trehalose accumulation were observed in conditions of heat stress. These results expand our understanding of the molecular mechanisms of the heat stress response of conidia and provide a foundation for future investigations.

[1]  S. Ying,et al.  A conidial protein (CP15) of Beauveria bassiana contributes to the conidial tolerance of the entomopathogenic fungus to thermal and oxidative stresses , 2011, Applied Microbiology and Biotechnology.

[2]  R. Dean,et al.  In-depth analysis of the Magnaporthe oryzae conidial proteome. , 2012, Journal of proteome research.

[3]  S. Lindquist,et al.  The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. , 1993, Annual review of genetics.

[4]  Proteins differentially expressed in conidia and mycelia of the entomopathogenic fungus Metarhizium anisopliae sensu stricto. , 2013, Canadian journal of microbiology.

[5]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[6]  A. Ram,et al.  Autophagy promotes survival in aging submerged cultures of the filamentous fungus Aspergillus niger , 2013, Applied Microbiology and Biotechnology.

[7]  M. Hahn,et al.  Trehalose metabolism is important for heat stress tolerance and spore germination of Botrytis cinerea. , 2006, Microbiology.

[8]  Chengshu Wang,et al.  Linkage of autophagy to fungal development, lipid storage and virulence in Metarhizium robertsii , 2013, Autophagy.

[9]  Kui Lin,et al.  RNA-Seq improves annotation of protein-coding genes in the cucumber genome , 2011, BMC Genomics.

[10]  J. Claverie,et al.  The significance of digital gene expression profiles. , 1997, Genome research.

[11]  É. Fernandes,et al.  Perspectives on the potential of entomopathogenic fungi in biological control of ticks. , 2012, Experimental parasitology.

[12]  P Ruoff,et al.  The Goodwin model: simulating the effect of cycloheximide and heat shock on the sporulation rhythm of Neurospora crassa. , 1999, Journal of theoretical biology.

[13]  J. Adams,et al.  The proteasome: structure, function, and role in the cell. , 2003, Cancer treatment reviews.

[14]  D. Kültz,et al.  Molecular and evolutionary basis of the cellular stress response. , 2005, Annual review of physiology.

[15]  A. Kassa,et al.  Production of thermotolerant entomopathogenic fungal conidia on millet grain , 2011, Journal of Industrial Microbiology & Biotechnology.

[16]  Siu-Ming Yiu,et al.  SOAP2: an improved ultrafast tool for short read alignment , 2009, Bioinform..

[17]  A. Beauvais,et al.  The diverse applications of RNA‐seq for functional genomic studies in Aspergillus fumigatus , 2012, Annals of the New York Academy of Sciences.

[18]  Lin Fang,et al.  WEGO: a web tool for plotting GO annotations , 2006, Nucleic Acids Res..

[19]  Ming Zhao,et al.  Transcriptomic profiling of Aspergillus flavus in response to 5-azacytidine. , 2013, Fungal genetics and biology : FG & B.

[20]  S. Gutiérrez,et al.  The heterologous overexpression of hsp23, a small heat-shock protein gene from Trichoderma virens, confers thermotolerance to T. harzianum , 2007, Current Genetics.

[21]  Harvey T. McMahon,et al.  Molecular mechanism and physiological functions of clathrin-mediated endocytosis , 2011, Nature Reviews Molecular Cell Biology.

[22]  I. Nookaew,et al.  A comprehensive comparison of RNA-Seq-based transcriptome analysis from reads to differential gene expression and cross-comparison with microarrays: a case study in Saccharomyces cerevisiae , 2012, Nucleic acids research.

[23]  J. Rosa,et al.  A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum. , 2010, Fungal biology.

[24]  M. Blythe,et al.  Genome-wide transcriptional response of Trichoderma reesei to lignocellulose using RNA sequencing and comparison with Aspergillus niger , 2013, BMC Genomics.

[25]  M. Gerstein,et al.  RNA-Seq: a revolutionary tool for transcriptomics , 2009, Nature Reviews Genetics.

[26]  J. Latgé,et al.  Transcriptomic analysis of the exit from dormancy of Aspergillus fumigatus conidia , 2008, BMC Genomics.

[27]  J. Walstad,et al.  Effects of environmental conditions on two species of muscardine fungi (Beauveria bassiana and Metarrhizium anisopliae) , 1970 .

[28]  Self-eating to grow and kill: autophagy in filamentous ascomycetes , 2013, Applied Microbiology and Biotechnology.

[29]  Chengshu Wang,et al.  Genetic engineering of fungal biocontrol agents to achieve greater efficacy against insect pests , 2009, Applied Microbiology and Biotechnology.

[30]  Yuxian Xia,et al.  Genetically altering the expression of neutral trehalase gene affects conidiospore thermotolerance of the entomopathogenic fungus Metarhizium acridum , 2011, BMC Microbiology.

[31]  Matthew D. Young,et al.  From RNA-seq reads to differential expression results , 2010, Genome Biology.

[32]  Hao Wang,et al.  Gene Expression Profiling of Clostridium botulinum under Heat Shock Stress , 2013, BioMed research international.

[33]  D. Rubinsztein,et al.  A novel link between autophagy and the ubiquitin-proteasome system , 2009, Autophagy.

[34]  B. Williams,et al.  Mapping and quantifying mammalian transcriptomes by RNA-Seq , 2008, Nature Methods.

[35]  M Marsh,et al.  The structural era of endocytosis. , 1999, Science.

[36]  Guo-Ping Zhao,et al.  Genome Sequencing and Comparative Transcriptomics of the Model Entomopathogenic Fungi Metarhizium anisopliae and M. acridum , 2011, PLoS genetics.

[37]  Hong Liu,et al.  Role of Trehalose Biosynthesis in Aspergillus fumigatus Development, Stress Response, and Virulence , 2010, Infection and Immunity.

[38]  Shizhu Zhang,et al.  Contribution of the gas1 Gene of the Entomopathogenic Fungus Beauveria bassiana, Encoding a Putative Glycosylphosphatidylinositol-Anchored β-1,3-Glucanosyltransferase, to Conidial Thermotolerance and Virulence , 2011, Applied and Environmental Microbiology.

[39]  Hsiao-ling Lu,et al.  Overexpression of a Metarhizium robertsii HSP25 gene increases thermotolerance and survival in soil , 2013, Applied Microbiology and Biotechnology.

[40]  Judith K Pollack,et al.  Autophagy in filamentous fungi. , 2009, Fungal genetics and biology : FG & B.

[41]  D. Rangel,et al.  Effects of physical and nutritional stress conditions during mycelial growth on conidial germination speed, adhesion to host cuticle, and virulence of Metarhizium anisopliae, an entomopathogenic fungus. , 2008, Mycological research.

[42]  A. Masuda,et al.  In vitro assessment of Metarhizium anisopliae isolates to control the cattle tick Boophilus microplus. , 2000, Veterinary parasitology.

[43]  Yoshihiro Yamanishi,et al.  KEGG for linking genomes to life and the environment , 2007, Nucleic Acids Res..

[44]  R. Brambl,et al.  Heat shock response of Neurospora crassa: protein synthesis and induced thermotolerance , 1985, Journal of bacteriology.

[45]  H. Stenmark,et al.  Mechanisms and functions of endocytosis , 2008, The Journal of cell biology.

[46]  M. Bidochka,et al.  Expression of genes involved in germination, conidiogenesis and pathogenesis in Metarhizium anisopliae using quantitative real-time RT-PCR. , 2006, Mycological research.

[47]  N. Talbot,et al.  Genome-wide Transcriptional Profiling of Appressorium Development by the Rice Blast Fungus Magnaporthe oryzae , 2012, PLoS pathogens.

[48]  J. Lord From Metchnikoff to Monsanto and beyond: the path of microbial control. , 2005, Journal of invertebrate pathology.

[49]  Reinhard Guthke,et al.  Integrative analysis of the heat shock response in Aspergillus fumigatus , 2010, BMC Genomics.

[50]  F. Shang,et al.  Ubiquitin-proteasome pathway and cellular responses to oxidative stress. , 2011, Free radical biology & medicine.

[51]  B. Bukau,et al.  Coordination of Translational Control and Protein Homeostasis during Severe Heat Stress , 2013, Current Biology.

[52]  A. Anderson,et al.  Mutants and isolates of Metarhizium anisopliae are diverse in their relationships between conidial pigmentation and stress tolerance. , 2006, Journal of invertebrate pathology.

[53]  S. Ryter,et al.  Autophagy: An Integral Component of the Mammalian Stress Response. , 2013, Journal of biochemical and pharmacological research.