Laser microdissection of grapevine leaves reveals site-specific regulation of transcriptional response to Plasmopara viticola

Grapevine (Vitis vinifera) is one of the most important fruit crops in the world and it is highly susceptible to downy mildew, caused by the biotrophic oomycete Plasmopara viticola. Gene expression profiling has largely been used to investigate regulation processes of grapevine-P. viticola interaction, but all studies have involved the use of whole leaves. However, only a small fraction of host cells is in contact with the pathogen and highly localized transcriptional changes of infected cells may be masked by the large portion of not-infected cells when analysing the whole leaf. In order to characterise the transcriptional regulation of the plant reaction with spatial resolution, we optimized a laser microdissection protocol to harvest stomata and surrounding cells from leaves of in vitro-grown grapevines at early stages of P. viticola infection. The expression levels of seven P. viticola responsive genes were greater in microdissected cells than in whole leaves, highlighting the site-specific transcriptional regulation of the host response. The gene modulation was restricted to the stomata cells and to the surrounding areas of infected tissues, indicating that short-distance signals are implicated. The high sensitivity of the laser microdissection analysis showed significant modulations of three genes that were completely masked in the whole tissue analysis. These results highlight that the transcriptional regulation of the host response to a biotrophic pathogen is mainly located to the infection sites. The optimized protocol for the laser microdissection analysis is suitable to increase the sensitivity of further high-throughput transcriptomic studies of the grapevine-P. viticola interaction