acridum but does not affect its virulence. The use of the RNAi mutant of Ntl could provide a new strategy for improving the conidiospore thermotolerance of an entomopathogenic fungus without compromising its virulence. Methods Strain growth conditions M. acridum strain CQMa102, a locust-specific strain, was isolated by our laboratory in Chongqing, China. Conidia were harvested from cultures grown on 1/4 strength Sabouraud’s dextrose agar medium (SDA: 1% dextrose, 0.25% mycological peptone, 2% agar, and 0.5% yeast BYL719 molecular weight extract) at 28°C. Mycelia for DNA and RNA extraction were grown by inoculating 100 mL 1/4 SDA liquid media with 106 conidia and incubating at 28°C with shaking at 150 rpm for 2-3 days. Construction of the Ntl over-expression

vector An over-expression vector (pBarEx) for filamentous fungi was constructed based on pBTM. pBarEx contained a bar gene, PD-0332991 mouse promoter pGpdA, and terminator TTrpC from A. nidulans and a polylinker between pGpdA and TTrpC. The full cDNA sequence of Ntl was amplified using Pyrobest DNA polymerase (TaKaRa, Quisinostat purchase Japan) with primers B1 (5′-AAT TAC GCG TAC CTC CAC GTT CGT CAG TC-3′ with an MluI recognition sequence at the 5′ end) and B2 (5′-CGC CAC GCG TTT GAG AGG GCA ATT AAT CG-3′ with an MluI recognition sequence at the 3′ end). The PCR product and vector pBarEx were both digested with MluI, and then ligated using T4 DNA ligase (pBarEx-NTL, Figure 1A). Construction of the Ntl RNAi vector

A dual promoter RNAi vector for filamentous fungi was first constructed based on pBTM, which was reported previously [44], pDPB containing a selectable Adenosine marker, the bar gene (resistance to ammonium glufosinate), polylinker, and two promotors in opposite direction (pGpdA

and pTrpC from A. nidulans). A fragment of the coding sequence of Ntl (310-745) was then amplified from M. acridum Ntl cDNA with primers A1 (5′-ATT AAC GCG TAG CAC AAG AAG ATA CCG ATG-3′ with an MluI restriction site at the 5′ end) and A2 (5′-TAT AAC GCG TCG CGC CAG GGA GCT GCT GGA CAT CTAG-3′ with an MluI restriction site at the 3′ end), which was designed according to the CQMa102 Ntl cDNA sequence (GenBank AY557612). The PCR product and vector pDPB were both digested with MluI, and then ligated using T4 DNA ligase (Takara, Japan) (pDPB-NTL) (Figure 1B). Transformation of M. acridum Intact M. acridum CQMa102 conidia were transformed by microparticle bombardment (Model PDS-1000/He biolistic particle delivery system, Bio-Rad, USA). For bombardment, 50 μL of conidia suspension (109 conidia/mL) were placed in the center of a Petri dish. Plasmids pDPB-NTL and pBarEx-NTL were linearized with BamHI and bound to 0.6-μm diameter golden particles and then transformed into M. anisoplia by particle-mediated DNA delivery (Model PDS-1000/He biolistic particle delivery system, Bio-Rad, USA), according to St Leger [45]. Following bombardment, conidia were resuspended in 5 mL of MilliQ water. Aliquots of 200 μL were plated on Czapek’s medium (3% saccharose, 0.