AA-ending codon-based computational analyses uncover novel cytoplasmic effectors in the Magnaporthe oryzae secretome

Phytopathogens secrete diverse, sequence-unrelated effector proteins outside (apoplastic) or into (cytoplasmic) plant cells to suppress host defenses and cause devastating diseases. Effectors rapidly evolve to avoid counter-detection by the host, and fungal cytoplasmic effectors lack recognizable host cell-targeting motifs, impeding cataloguing of the full suite of host-deployed effectors encoded in pathogen genomes. In the devastating rice blast fungus Magnaporthe oryzae , cytoplasmic and apoplastic effectors are secreted by different routes, but candidates must be confirmed by fluorescent labelling, consequently few are known. AA-ending codon usage rates are the fraction of AA-ending codons as a percentage of the total number of AA- and synonymous AG-ending codons in an mRNA. In M. oryzae , high AA % rates of the corresponding mRNAs distinguished seven experimentally confirmed cytoplasmic effectors from four experimentally confirmed apoplastic effectors, but whether AA % rates can identify new effectors is unknown. Here, using computational analyses and live-cell imaging confirmation, we successfully predicted, based on AA % rates, two new cytoplasmic effectors and two apoplastic effectors in the M. oryzae secretome. Our findings support the notion that cytoplasmic effector mRNAs are globally enriched for AA-ending codons, aiding effector discovery and the search for novel sources of durable crop resistance.