Suppression of the pathogen-inducible Medicago truncatula putative protease-inhibitor MtTi2 does not influence root infection by Aphanomyces euteiches but results in transcriptional changes from wildtype roots

Abstract During the parasitic interaction between the model legume Medicago truncatula and the oomycete Aphanomyces euteiches , a plant protease inhibitor (PI)-encoding gene, MtTi 2, was found to be induced. Sequence and database analyses showed that MtTi 2 belongs to a group of at least four PIs, which are all specifically expressed upon pathogen infection or elicitor treatment. Reporter analysis showed that the MtTi 2 promoter is activated in infected root tissues and revealed a distinct promoter area involved in this transcriptional induction. In order to elucidate the function of MtTi 2, RNA i -mediated silencing experiments were carried out. After introduction of double-stranded MtTi 2 RNA into M. truncatula roots, no endogenous transcripts were observed, indicating efficient gene suppression. Since some plant protease inhibitors were shown to suppress pathogen growth, the MtTi 2 i -phenotype was analyzed with respect to pathogen spreading after root infection. No altered pathogen development was observed in MtTi 2i-roots as compared to control roots, indicating that MtTi 2 does not directly influence the pathogen. In order to investigate whether suppression of MtTi 2 results in transcriptional changes, transcriptome profiles of MtTi 2 i roots and control roots were analyzed using an 8000 gene M. truncatula microarray. A cluster analysis of genes regulated in roots upon infection in two MtTi 2 i -lines or two vector lines revealed groups of genes showing different regulation in MtTi 2 i - and wild type roots. Hence, it is likely that MtTi 2 has endogenous functions other than directly suppressing the pathogen. Furthermore, the transcriptome approach revealed insight into the transcriptional changes in response to A. euteiches infection.