Determination of fungal gene expression in planta by qRT-PCR and characterization of putative pathogenicity related genes of Verticillium longisporum

Abstract

Genes of <i>Verticillium longisporum</i> potentially involved in the pathogenicity of the fungus were identified in previous studies. The purpose of this work was to characterize some of these genes. Short transcript-derived fragment obtained after differential cDNA-AFLP were used as the starting material. Full-length sequences were obtained using RACE-PCR (rapid amplification of cDNA-ends with polymerase chain reaction), inverse PCR on self circularized genomic DNA, by screening of a genomic library and by cloning PCR products generated with primers based on published sequences from the genome of <i>V. dahliae</i>. A genomic library of <i>Verticillium longisporum</i> was constructed, consisting of about 10,000 plasmids arrayed in 96-wells microplates. The insert size was 8-12 kb. The library represented approximately 50% of the genome. The library was hierarchically ordered and pools of whole microplates, rows and columns were generated. These pools allowed the identification of a single sequence in the library by PCR with less than 40 PCR reactions. Full-length genomic copies of several candidate genes were obtained in this way. Analysis of the expression of candidate genes <i>in planta</i> by real-time RT-PCR was established. Fungus colonizing the root and shoot tissue of infected <i>Brassica napus</i> plants was analyzed separately. The isolation, purification and transcription procedures of total RNA and mRNA from <i>V. longisporum</i> infected <i>Brassica</i> plants were improved and used for the determination of relative gene expression by qRT-PCR. A set of reference genes were tested. The <i>in planta</i> determination of relative gene expression of pathogenicity related genes in <i>V. longisporum</i> compared to growth <i>in vitro</i> in a xylem-simulating medium (SXM) by quantitative reverse transcription real-time PCR (qRT-PCR) was applied for description of TDFs after cDNA-AFLP screening and sequence extensions. 10 genes have been characterised after their relative expression levels <i>in planta</i> by qRT-PCR, 6 of them indicated an up-regulated gene expression, 2 of them were down-regulated and further 2 were time-depended. One of the candidate genes, designated as Vl_6.2, was identified in previous work due to strong induction of its expression by xylem sap metabolites of <i>B. napus</i>. This finding was confirmed by transcript analysis <i>in planta</i>. Vl_12.1 gene, its gene expression was reduced during infection of <i>B. napus</i> in root/hypocotyl tissue in reference to <i>in vitro</i> grown mycelium in a xylem simulating artificial medium, indicating a suppression of this gene during the infection process. The gene has an ORF of 2,328 nucleotides, one intron and translated cDNA is predicted to code for a 775 amino acids. Sequence analysis of Vl_12.1 showed the high homology to the zinc-finger transcription factor ACE1 containing three ZnF_C2H2 domains. We applied RNAi technology for gene silencing of this ACE1-like Vl_12.1 gene in <i>V. longisporum</i> by expressing a gene-specific RNA-hairpin. Gene-silenced mutants did not show any visual difference in triggering typical infection symptoms in <i>B. napus</i>. A semi-quantitative testing for alterations in cellulase activities on cellulose-containing agar medium showed less difference between the silenced mutants and <i>V. longisporum</i> wild type.