The M agnaporthe oryzae effector AVR 1– CO 39 is translocated into rice cells independently of a fungal‐derived machinery

Summary Effector proteins are key elements in plant–fungal interactions. The rice blast fungus M agnaporthe oryzae secretes numerous effectors that are 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 ( ORF 3 ) corresponding to the M . oryzae avirulence gene AVR 1– CO 39 that interacts with the rice resistance gene P i– CO 39 and encodes a small secreted protein without homology to other proteins. We demonstrate that AVR 1– CO 39 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 AVR 1– CO 39 and the monomeric red fluorescent protein ( mRFP ) indicated that AVR 1– CO 39 is translocated into the cytoplasm of infected rice cells. Transient expression of an AVR 1– CO 39 isoform without a signal peptide in rice protoplasts triggers a P i– CO 39‐ specific hypersensitive response, suggesting that recognition of AVR 1– CO 39 by the P i– CO 39 gene product occurs in the cytoplasm of rice cells. The native AVR 1– CO 39 protein enters the secretory pathway of rice protoplasts as demonstrated by the ER localization of AVR 1– CO 39: mRFP : HDEL translational fusions, and is correctly processed as shown by W estern blotting. However, this secreted AVR 1– CO 39 isoform triggers a P i– CO 39 ‐specific hypersensitive response and accumulates inside rice protoplasts as shown by W estern blotting and localization of AVR 1– CO 39: mRFP translational fusions. This indicates that AVR 1– CO 39 is secreted by rice protoplasts and re‐enters into the cytoplasm by unknown mechanisms, suggesting that translocation of AVR 1– CO 39 into rice cells occurs independently of fungal factors.