Phire plant direct pcr

A PCR fragment was amplified from FGSC 2489 genomic DNA with the primers NCU00378_XbaI_F and NCU00378_PacI_R (Additional file 1: Table S8) using Phusion DNA polymerase (NEB # M0530). The final product was subjected to PCR purification with Qiaquick PCR kit (Catalog # 28104) and cloned into the pCR^® vector (Invitrogen # 44-0302) according to the manufacturer’s protocol. The NCU00378 insert was excised from the vector with XbaI and PacI and cloned into a modified pMF272 vector cut with XbaI/PacI in frame with an eGFP reporter ORF and flanked by tef-I promoter and ccg-1 terminator sequences from pMF272 [72 (link)]. The deletion strains for NCU00378 (FGSC 12919) were crossed with FGSC 6103 to create an NCU00378 deletion in a his3⁻ background. Progeny were genotyped by Phire Plant Direct PCR (Thermo Scientific # F-130WH) on conidia with the primers Hphp and NCU00378_3r (Additional file 1: Table S8).
Transformation was performed by electroporation according to a protocol available at the FGSC or as modified by Ziv and Yarden [73 (link)]. Conidia from the selected transformants were genotyped by PCR for the hygromycin cassette and also for the NCU00378-GFP construct with the primers NCU00378_F and pMF272_Rev (Additional file 1: Table S8).

Protoplasts from the isolate Guy11 of P. oryzae were transformed by heat shock with 10μg of KpnI-linearized plasmids for the expression of MAX effectors or RFP as described previously [88 (link)]. After two rounds of antibiotic selection and isolation of monospores, transformed isolates were genotyped by Phire Plant Direct PCR (Thermo Scientific) using primers described in S7 Table. The Guy11 transgenic isolates expressing AVR-Pia and AVR1-CO39 were previously generated [50 (link), 89 (link)].