Laser microdissection of grapevine leaves highlights site-specific transcriptional changes at the early stages of downy mildew infection

Grapevine (Vitis vinifera) is one of the world’s major fruit crops, but most of the commercial cultivars are susceptible to downy mildew, caused by Plasmopara viticola. Transcript profiling has largely been used to investigate gene expression changes of the interaction between grapevine and P. viticola, but these studies have generally involved the use of RNA from whole grapevine leaves. P. viticola infects grapevine leaves and young berries by stomata and develops intercellular mycelium in the mesophyll. Only a small fraction of leaf cells is in contact with the pathogen at the early stages of infection and the large portion of not-infected cells could mask the transcriptional changes related to defence activation. Laser microdissection (LMD) technique allows the isolation of specific cell types from heterogeneous tissue. LMD was used to specifically collect cells at the site of P. viticola infection or at the adjacent layers from inoculated leaves of in vitro-grown grapevines. Protocols for sample fixation, laser microdissection and RNA isolation from group of cells were optimized and the expression of ten genes involved in the grapevine defence response was analysed by real-time RT-PCR. The expression level of the selected genes was generally greater at the site of infection compared to the whole infected leaf, and expression profiles in infected and adjacent cells differed according to the tested genes. These results get new insights on the activation of specific processes at the sites of P. viticola infection, which were masked in the whole-leaf analysis, and the optimized protocols will be further used for site-specific transcriptomic studies