Nicotiana benthamiana γ-Thionin Synthesis Is Induced in Response to Foreign Nucleus-Targeted Proteins

Pathogenic and symbiotic bacteria secret protein factors to affect the host cell nucleus by directly inducing chromatin remodeling, interfering with transcription, modulating DNA replication and mRNA synthesis. These bacterial molecules are called nucleomodulins. During evolution, plants acquired a great variety of defense mechanisms including the synthesis of such antimicrobial peptides (AMPs) as defensins. We have demonstrated that transient production of a foreign protein containing nuclear localization signal (NLS) in Nicotiana benthamiana leaves leads to the induction of the γ-thionin (NbγThio) mRNA accumulation. We hypothesized that γ-thionin that belongs to the defensin group of AMPs is induced by nucleomodulins of pathogenic bacteria and in particular in response to their NLSs. To check this suggestion, we used GFP fused to NLS from Agrobacterium tumefaciens VirE3 protein and to a model NLS from human prothymosin α. Both NLSs appeared to be functionally active and target GFP to the nucleus of N. benthamiana cells in a transient expression system. Moreover, they induced synthesis of NbγThio mRNA. We isolated NbγThio promoter (Pr-γThio) and obtained a genetic construct encoding a reporter protein β-glucuronidase (GUS) under control of Pr-γThio for expression in N. benthamiana. Using Pr-γThio:GUS vector we further demonstrated that Pr-γThio is sensitive to the synthesis of foreign NLS-containing proteins: GUS-encoding mRNA level was significantly higher when Pr-γThio:GUS was co-expressed with 35S:GFP_NLS. Thus, we have shown that NbγThio gene expression is activated in response to bacterial nucleus-targeted proteins in the cell via induction of Pr-γThio. This research was funded by the Russian Science Foundation (project No. 19-74-20031) and work on promoter isolation was supported by Russian Foundation for Basic Research (project No. 17-29-08012).