SERRALYSIN-LIKE PROTEIN OF Candidatus liberibacter asiaticus AFECTS EXTRACELULAR MATRIX IN HETEROLOGOUS SYSTEMS AND VIRULENCE IN HOST PLANTS

BRUNA, R.; GONZALEZ, C; MOLINA, MC; TORRES, P; GADEA, J; GARCÍA, R; GARCÍA VÉSCOVI, ELEONORA

Virtual

Congreso; LVI SAIB Meeting ? XV SAMIGE Meeting; 2020

SAIB-SAMIGE (Congreso conjunto)

Huanglongbing (HLB) is the major threat for Citrus species and it is caused by intracellular alfa-proteobacteria belong to the genus Candidatus Liberibacter (CaL), being ´Ca. L. asiaticus´ (Las) the prevalent specie. This bacterium is psyllid-transmitted and resides within the citrus phloem. Infected trees symptoms -blotchy and mottled leaves, small fruits, as well as shorter lifespan and premature fruit drop- have been associated with an increase of callose deposition and starch accumulation that constricts symplastic transport producing a source-sink imbalance. The inability to culture CaL has hampered progress in studying the pathogen and its virulence mechanisms, our limited understanding relies on transcriptome and proteomic data and on the expression of putative virulence proteins in surrogate models. Those studies highlight the idea that Las actively manipulate plant defense response. Extracellular proteases which belong to the Serralysin metalloprotease family are wide known virulence factors exported through the secretion system type I (SST1). A putative serralysin gene was identified in CaL genomes and its expression levels was increased when Las changed it environment from the psyllid to the citrus leaf, suggesting a function in the infection process. Here, we study the function of the Las-serralysin (hereafter Las_1345) as virulence factor. First, we decipher whether Las_1345 could be secreted through bacteria that express SST1-secreted proteases such as Serratia marcescens (Smc) and Xanthomonas citri and X. campestris (Xac and Xcc, respectively). Las_1345 only was detected in the pellet fraction of every surrogate bacteria, nevertheless, we measured intracellular protease activity. Las_1345 expression increased the proteolityc activity of Xcc by more than 50% and partially restored (5?10%) the protease activity of protease deficient Smc strain (prtA). Las_1345 purified from E. coli also showed low proteolytic activity. Then, we analyzed whether Las_1345 can exert its activity on plant proteins. Las_1345-GFP was localized at the cell membrane in Nicotiana benthamiana and tissue integrity was not affected. Moreover, protease activity measured in tissue cells expressing Las_1345 arose similar levels as in control tissue, suggesting that Las_1345 may not act as protease in plant tissue. Interesting, Las_1345 modify biofilm structure in surrogate bacteria, which was associated with less virulence in citrus plants. We propose that Las_1345 is a virulence factor contributing with Las colonization in the citrus phloem.