Homologues of the RNA binding protein RsmA in Pseudomonas syringae pv. tomato DC3000 exhibit distinct binding affinities with non‐coding small RNAs and have distinct roles in virulence

Abstract

Summary Pseudomonas syringae pv . tomato DC3000 ( Pst DC3000) contains five RsmA protein homologues. In this study, four were functionally characterized, with a focus on RsmA2, RsmA3 and RsmA4. RNA electrophoretic mobility shift assays demonstrated that RsmA1 and RsmA4 exhibited similar low binding affinities to non‐coding small RNAs (ncsRNAs), whereas RsmA2 and RsmA3 exhibited similar, but much higher, binding affinities to ncsRNAs. Our results showed that both RsmA2 and RsmA3 were required for disease symptom development and bacterial growth in planta by significantly affecting virulence gene expression. All four RsmA proteins, especially RsmA2 and RsmA3, influenced γ‐amino butyric acid utilization and pyoverdine production to some degree, whereas RsmA2, RsmA3 and RsmA4 influenced protease activities. A single RsmA, RsmA3, played a dominant role in regulating motility. Furthermore, reverse transcription quantitative real‐time PCR and western blot results showed that RsmA proteins, especially RsmA2 and RsmA3, regulated target genes and possibly other RsmA proteins at both transcriptional and translational levels. These results indicate that RsmA proteins in Pst DC3000 exhibit distinct binding affinities to ncsRNAs and have distinct roles in virulence. Our results also suggest that RsmA proteins in Pst DC3000 interact with each other, where RsmA2 and RsmA3 play a major role in regulating various functions in a complex manner.

Two Homologues of the Global Regulator Csr/Rsm Redundantly Control Phaseolotoxin Biosynthesis and Virulence in the Plant Pathogen Pseudomonas amygdali pv. phaseolicola 1448A

“…This type of partial complementation, or complementation to the reverse phenotype, has already been reported for rsm homologues in P. syringae pv. tomato DC3000 [ 9 , 10 ], again suggesting the relevance of the relative concentration of Rsm proteins in the cell.…”

“…Homologues of RsmA (CsrA2) and RsmE (CsrA3) are present in many, but not all bacteria containing a Csr/Rsm system; members of the other Rsm subfamilies, however, have a rather patchy distribution [ 2 ]. Although the exact role of the different homologues is often unclear, some of them were shown to be redundant and/or have unique regulatory roles, which likely provide genetic flexibility and helps to fine-tune the regulatory responses [ 6 , 7 , 8 , 9 , 10 ].…”

“…There is no correlation between the numbers of sRNAs and Csr/Rsm homologues in a given genome and, likewise, the sRNAs do not appear to be specific for each Csr/Rsm homologue [ 5 ]. Nevertheless, the sRNAs contribute to a differential control of the Csr/Rsm system because they show distinct affinities for different Csr/Rsm homologues and because they have distinct expression patterns, which can also change between different bacteria [ 10 , 14 , 15 ].…”

“…P. syringae pv. tomato DC3000 contains five csr / rsm genes [ 9 ], and they were shown to contribute to the regulation of relevant roles for the bacterial life cycle, including the interaction with the plant host [ 9 , 10 ]. Homologues rsmA and rsmE (also called csrA2 / rsmA2 and csrA3 / rsmA3 , respectively) acted both individually and synergistically for the control of diverse phenotypes, displaying certain regulatory specificities, whereas no major roles could be assigned to the other homologues.…”

Comparative transcriptomic analysis of global gene expression mediated by (p) ppGpp reveals common regulatory networks in Pseudomonas syringae

“…PssB728a ) [26,27] were routinely cultured on King's medium B (KB) plate, or 5 ml of fresh KB broth at 28°C with shaking at 250 rpm. The hrp-inducing medium (HMM) supplemented with 10 mM fructose as carbon source was used for RNA isolation [26,27,107]. Bacterial growth was monitored by measuring absorbance of cell suspensions at 600 nm.…”