Updated project metadata. “Inaccurate” expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence indicates that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering new capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism. In this study, we address this problem by studying duplicated genes of aminoacyl-tRNA synthetases. Using bacterial prolyl-tRNA synthe¬tase (ProRS) genes as an example, we identify an anomalous ProRS isoform, ProRSx, and a corre¬sponding tRNA, tRNAProA, that are predominately found in plant pathogens from Streptomyces species. We then show that tRNAProA has an unusual hybrid structure that allows this tRNA to mistranslate alanine codons as proline. We finally provide biochemical, genetic, and mass-spectro-metric evidence that cells which express ProRSx and tRNAProA can translate GCU alanine codons both as alanine and proline. This dual use of alanine codons creates a hidden proteome diversity due to stochastic Ala→Pro mutations in protein sequences. Thus, we show that important plant pathogens are equipped with a tool to alter the identity of their sense codons. This finding reveals the first example of a natural tRNA synthetase/tRNA pair for dedicated mistranslation of sense codons.