Unconventional secretion of Magnaporthe oryzae effectors in rice cells is regulated by tRNA modification and codon...

Microbial pathogens deploy effector proteins to manipulate host cell innate immunity, often using poorly understood unconventional secretion routes. Transfer RNA (tRNA) anticodon modifications are universal, but few biological functions are known. Here, in the rice blast fungus Magnaporthe oryzae, we show how unconventional effector secretion depends on tRNA modification and codon usage. We characterized the M. oryzae Uba4-Urm1 sulfur relay system mediating tRNA anticodon wobble uridine 2-thiolation (s²U₃₄), a conserved modification required for efficient decoding of AA-ending cognate codons. Loss of s²U₃₄ abolished the translation of AA-ending codon-rich messenger RNAs encoding unconventionally secreted cytoplasmic effectors, but mRNAs encoding endoplasmic reticulum-Golgi-secreted apoplastic effectors were unaffected. Increasing near-cognate tRNA acceptance, or synonymous AA- to AG-ending codon changes in PWL2, remediated cytoplasmic effector production in Δuba4. In UBA4⁺, expressing recoded PWL2 caused Pwl2 super-secretion that destabilized the host-fungus interface. Thus, U₃₄ thiolation and codon usage tune pathogen unconventional effector secretion in host rice cells.