Effector proteins are key elements in plant fungal interactions. The rice blast fungus Magnaporthe oryzae secretes numerous effectors suspected to be translocated inside plant cells. However, their cellular targets and the mechanisms of translocation are still unknown. Here, we have identified the open reading frame (ORF3) corresponding to the M. oryzae avirulence gene AVR1-C039 interacting with the rice resistance gene Pi-CO39 and encoding a small secreted protein without homologies to other proteins. We demonstrate that AVR1-C039 is specifically expressed and secreted at the plant fungal interface during the biotrophic phase of infection. Live cell imaging with M. oryzae transformants expressing a translational fusion between AVR1-C039 and the monomeric red fluorescent protein (mRFP), indicates that AVR1-C039 is translocated into the cytoplasm of infected rice cells. Transient expression of an AVR1-C039 isoform without signal peptide in rice protoplasts triggers a Pi-C039 specific HR suggesting that the recognition of AVR1-C039 by the Pi-C039 gene product occurs in the cytoplasm of rice cells. The native AVR1-C039 enters into the secretory pathway of rice protoplasts as demonstrated by the ER localization of AVR1-CO39:mRFP:HDEL translational fusions and is correctly processed as shown by Western blotting. However, this secreted AVR1-C039 isoform triggers Pi-CO39 specific HR and accumulates inside rice protoplasts as shown by western blotting and localisation of AVR1-C039:mRFP translational fusions. This indicates that AVR1-C039 is secreted by rice protoplasts and re-enters into the cytoplasm by unknown mechanisms suggesting that translocation of AVR1-C039 into rice cells occurs independently of fungal factors.
Cécile Ribot, Stella Césari, Imène Abidi, Véronique Chalvon, Caroline Bournaud, Julie Vallet, Marc-Henri Lebrun, Jean-Benoit Morel and Thomas Kroj