Bennypaul, Harvinder

ABSTRACT X‐disease, caused by strains in the 16SrIII‐A subgroup of ‘ Candidatus Phytoplasma pruni’, is a devastating disease of Prunus species (stone fruits). Multiple outbreaks of this disease have occurred across much of North America for more than a century, with the most recent one beginning around 2010 in the Pacific Northwest of the United States, causing severe damage to the stone fruit industry. Sensitive and specific detection of X‐disease is critical to prevent the propagation and spread of infected plant material; however, current PCR‐based detection workflows lack specificity to X‐disease and require one or more PCR assays followed by additional testing to confirm the species identity. In this study, two real‐time PCR assays, one targeting the secY gene and the other targeting the tuf gene, were developed for specific detection of X‐disease in stone fruits. The CPH‐TXD assay targeting the tuf gene provided high‐sensitivity target detection of as few as five target copies with significantly reduced cross‐reactivity compared to current methods. The CPH‐SXD assay targeting the secY gene detected a minimum of 30 target copies and showed no cross‐reaction with closely related strains. The repeatability and reproducibility of both assays were verified with acceptable results. Together, these assays provide efficient and high‐specificity detection methods to combat the growing threat of X‐disease in North America.

GroE is a chaperonin folding system consisting of GroEL (Cpn60, a 60 kDa chaperonin), and the smaller co-chaperonin GroES (Cpn10). Many “client” proteins require GroE to fold properly, including several that are essential for cell viability. Unsurprisingly then, GroE is found in nearly all bacteria and eukaryotes. Mollicutes are the only microorganisms that lack GroE in almost all cases. Only two clades of Mollicutes have retained the ancestral GroE system, or perhaps reacquired one; these exceptions include the family Acholeplasmataceae (consisting of the genera Acholeplasma and Phytoplasma). The role of GroEL in these “exceptional” Mollicutes is a source of speculation, given how many non-canonical “moonlighting” roles have been ascribed to this protein. GroEL has been suggested to play a role in pathogenesis in plant and animal pathogenic Mollicutes, by binding to host cells and facilitating invasion. However, in one further layer of exception, the phytopathogenic taxon ‘Candidatus Phytoplasma pruni’ (ribosomal group 16SrIII), was reported to lack a GroE system. This study confirms the lack of a functional GroE system in 16SrIII by providing two new, high quality, non-fragmented genome assemblies, as well as a thorough survey of other 16SrIII genomes for genes encoding GroEL/GroES, including those that may not resemble phytoplasma groEL (ie. acquired by horizontal gene transfer, HGT). We discuss the implications of a clearly phytopathogenic, invasive group of Mollicutes that nevertheless lacks GroE, in light of the presumed role of GroEL for these microorganisms. We determined that three groups of genomes of 16SrIII contain short, non-functional groEL pseudogenes, while most of the reported genomes lack any semblance of a GroE system. Examination of the new assemblies allowed us to rule out HGT as a means of GroE acquisition.