Exploring the expression and functionality of the rsm sRNAs in Pseudomonas syringae pv. tomato DC3000

Abstract

The Gac-rsm pathway is a global regulatory network that governs mayor lifestyle and metabolic changes in gamma-proteobacteria. In a previous study, we uncovered the role of CsrA proteins promoting growth and repressing motility, alginate production and virulence in the model phytopathogen Pseudomonas syringae pv. tomato (Pto) DC3000. Here, we focus on the expression and regulation of the rsm regulatory sRNAs, since Pto DC3000 exceptionally has seven variants (rsmX1-5, rsmY and rsmZ). The presented results offer further insights into the functioning of the complex Gac-rsm pathway and the interplay among its components. Overall, rsm expressions reach maximum levels at high cell densities, are unaffected by surface detection, and require GacA for full expression. The rsm levels of expression and GacA-dependence are determined by the sequences found in their −35/-10 promoter regions and GacA binding boxes, respectively. rsmX5 stands out for being the only rsm in Pto DC3000 whose high expression does not require GacA, constituting the main component of the total rsm pool in a gacA mutant. The deletion of rsmY and rsmZ had minor effects on Pto DC3000 motility and virulence phenotypes, indicating that rsmX1-5 can functionally replace them. On the other hand, rsmY or rsmZ overexpression in a gacA mutant did not revert its phenotype. Additionally, a negative feedback regulatory loop in which the CsrA3 protein promotes its own titration by increasing the levels of several rsm RNAs in a GacA-dependent manner has been disclosed as part of this work.

Distinctive features of the Gac‐Rsm pathway in plant‐associated Pseudomonas

“…Another conserved feature is the induction of the expression and/or activity of GacA/UvrY by the CsrA/Rsm proteins. This usually results in enhanced Csr/Rsm RNA expression (Gudapaty et al ., 2001; Suzuki et al ., 2002; Kay et al ., 2005; Fortune et al ., 2006; Humair et al ., 2010; Camacho et al ., 2015; Butz et al ., 2019; Ferreiro et al ., 2021), generating a negative feedback loop that reduces the response times to the stimuli (Adamson and Lim, 2013). Likewise, the CsrA/Rsm proteins would also affect the expression of other genes directly controlled by GacA/UvrY, if any.…”

“…Interestingly, P. syringae pv. tomato (Pto) DC3000 has seven Rsm RNAs (RsmX1‐5, RsmY and RsmZ) and the alignment of their promoter regions revealed that all of them have a likely GacA‐box (Moll et al ., 2010), but the motifs exhibit particularities which makes them to be differentially regulated by this protein (Ferreiro et al ., 2021). As it was mentioned before, the Rsm RNA levels are also controlled by other regulators, such as HptB and MvaT, which downregulate RsmZ expression in P. aeruginosa (Brencic et al ., 2009; Jean‐Pierre et al ., 2016), IHF, PsrA and AlgR, that promote the transcription of RsmZ in P. protegens CHA0 (Humair et al ., 2010; Li et al ., 2017), or a hairpin structure, which may be the target of an unknown transcriptional factor and is essential for full RsmZ expression in P. brassicacearum NFM421 (Lalaouna et al ., 2012a).…”

“…As it was mentioned before, the Rsm RNA levels are also controlled by other regulators, such as HptB and MvaT, which downregulate RsmZ expression in P. aeruginosa (Brencic et al ., 2009; Jean‐Pierre et al ., 2016), IHF, PsrA and AlgR, that promote the transcription of RsmZ in P. protegens CHA0 (Humair et al ., 2010; Li et al ., 2017), or a hairpin structure, which may be the target of an unknown transcriptional factor and is essential for full RsmZ expression in P. brassicacearum NFM421 (Lalaouna et al ., 2012a). Likewise, the P. protegens CHA0 RsmA and RsmE proteins and Pto DC3000 RsmE (CsrA3) exert a positive action on the transcription of the Rsm RNAs, which in turn results in a greater sequestration of them, generating a negatively feedback regulatory loop that will be discussed later (Kay et al ., 2005; Humair et al ., 2010; Sonnleitner and Haas, 2011; Ferreiro et al ., 2021).…”

“…We would like to emphasize the importance of studying the impact of all the factors that influence Rsm RNA expression, including GacA and other transcriptional regulators, as well as their stability and affinity for the different Rsm proteins. Also, it should be taken into account that low GacA‐dependent Rsm RNAs may be expressing at high levels in the gacA mutant, as it is the case of RsmX5 in Pto DC3000 (Ferreiro et al ., 2021) or RsmY in P. brassicacearum NFM421 (Lalaouna et al ., 2012b).…”

“…The complexity of the Gac‐Rsm system is even greater as the expression of the Rsm sRNAs significantly decreased when rsmA and/or rsmE were knocked out, and dramatically improved when overexpressed, indicating a feedback mechanism by which Rsm proteins regulate the level of its antagonistic sRNAs in a GacA‐dependent manner (Kay et al ., 2005; Humair et al ., 2010; Ferreiro et al ., 2021). RsmA and RsmE have this positive effect on P. protegens CHA0 RsmX, RsmY and RsmZ levels (Kay et al ., 2005; Humair et al ., 2010) and RsmE has been shown to increase RsmY, RsmZ and RsmX5 transcription in Pto DC3000 (Ferreiro et al ., 2021). In E. coli , this negative feedback has been shown to reduce response times since sequestration of CsrA by CsrB/C RNAs allows rapid reduction of CsrA activity without the need for its dilution via growth (Adamson and Lim, 2013).…”