Host-specific and homologous pairs of Melampsora larici - populina effectors unveil novel Nicotiana benthamiana stromule induction factors.

The poplar-rust fungus Melampsora larici-populina is part of one of the most devastating group of fungi (Pucciniales) and causes important economic losses to the poplar industry. As an heteroecious obligate biotroph, M. larici-populina spread depends on its ability to carry out its reproductive cycle through larch and then poplar parasitism. Genomic approaches have identified over a thousand candidate secreted effector proteins (CSEPs) from the predicted secretome of M. larici-populina which are potentially implicated in the infection process. In this study, we selected CSEP pairs (and one triplet) among CSEP gene families, which share high sequence homology but display specific gene expression profiles among the two distinct hosts. We determined their subcellular localization by confocal microscopy through expression in the heterologous plant system Nicotiana benthamiana. Five out of nine showed partial or complete chloroplastic localization. We also screened for potential protein interactors from larch and poplar by yeast two-hybrid. One pair of CSEPs and the triplet shared common interactors whereas the members of the two other pairs did not have common targets from either host. Finally, stromule induction quantification revealed that two pairs and the triplet of CSEPs induced stromules when transiently expressed in N. benthamiana. The use of N. benthamiana eds1 and nrg1 knock-out lines showed that CSEPs can induce stromules by an eds1-independent mechanism. However, CSEP homologs share the same impact on stromule induction and contributed to discovering a new stromule induction cascade that can be partially and/or fully independent of eds1.