Discovering clubroot resistance genes in Brassica vegetable crops

Clubroot, caused by the soil-borne obligate biotroph Plasmodiophora brassicae Woronin, is one of the most serious diseases of Brassica worldwide. In Australia, it is responsible for losses of at least 10% in crucifer yield causing more than AU$ 17 million in lost profits every year. To eliminate the use of expensive and environmentally harmful fungicides and to minimise loss, the breeding of resistant cultivars is an effective approach. The identification of the actual number of genes involved in clubroot resistance and their mechanisms of action could be important for effective breeding strategies.<br><br>This study documented the first report of the large-scale profiling of the transcriptional changes to the very early stages of P. brassicae infection in Brassica vegetable crops using a microarray approach. Firstly, a cost-effective ‘boutique’ Brassica oligonucleotide array of 150 Arabidopsis- / Brassica-derived features was constructed using nucleotide sequences from GenBank. This array, which was biased towards defence-associated and regulatory genes, was used to investigate the gene expressions of the partially-resistant Chinese cabbages ‘Tahono’ and ‘Leaguer’ and the susceptible ‘Granaat’ when challenged with aggressive Victorian clubroot isolate. The microarray data, validated by qRT-PCR, indicated a high number of constitutively and differentially expressed genes in response to pathogen attack, prominently at 48 hours after inoculation (hai). These responses may correlate with the timing of penetration by the primary zoospores of P. brassicae in the host root hair cells. The key observations were the constitutive over-expression and induction of a pathogenesis-related (PR) protein: chitinase as well as the up-regulation of a lignin biosynthesis enzyme: caffeoyl-CoA 3-O-methyltransferase in these partially-resistant varieties.<br><br>A more sophisticated microarray platform, the Affymetrix Arabidopsis ATH1 genechip, was used as an exploratory tool for whole-genome transcriptional profiling of ‘Tahono’ and ‘Granaat’ as well as a clubroot resistant fodder turnip ‘ECD04’ at 48 hai. A key observation was three major salicylic acid (SA)-dependent responses that were consistent with elevated level of SA in ‘Tahono’ and ‘ECD04’. SA possibly induced by the accumulation of reactive oxygen species, appears to be an essential regulatory component in firstly, the constitutive over-expression of the PR protein endochitinase, possibly via the NPR1, WRKY and TGA factors. Secondly, the high basal level of lignification via a peroxidase-dependent pathway in the roots provided an enhanced physical barrier against clubroot. Lastly, the myrosinase / glucosinolate defence system may be regulated via the SA signalling. The high basal level of myrosinase was probably involved in the rapid hydrolysis of aliphatic glucosinolates into toxic antifungal by-products while the low basal glucosinolate root content (possibly indole glucosinolate) may have resulted in less severe gall formation in the partially-resistant / resistant Brassica varieties.<br><br>These conclusions were based on previous reports on the putative role of these genes and further validation via other techniques such as SuperSAGE, ‘knock-out’ mutants or over-expressing transgenics, is required. Ultimately, these candidate genes involved in defence may potentially be used in the development of functional molecular markers for the marker-assisted selection of clubroot resistant Brassica vegetable crops<br>