The type 3 effector NopL ofSinorhizobiumsp. strain NGR234 is a mitogen-activated protein kinase substrate

Abstract

Pathogenic bacteria utilize type 3 secretion systems to inject type 3 effectors (T3Es) into host cells, thereby subverting host defense reactions. Similarly, T3Es of symbiotic nitrogen-fixing rhizobia can affect nodule formation on roots of legumes. Previous work showed that NopL (nodulation outer protein L) of Sinorhizobium(Ensifer) sp. strain NGR234 is multiply phosphorylated in eukaryotic cells and that this T3E suppresses responses mediated by mitogen-activated protein (MAP) kinase signaling in yeast (mating pheromone signaling) and plant cells (expression of pathogenesis-related defense proteins). Here, we show that NopL is a MAP kinase substrate. Microscopic observations of fluorescent fusion proteins and bimolecular fluorescence complementation analysis in onion cells indicated that NopL is targeted to the nucleus and forms a complex with SIPK (salicylic acid-induced protein kinase), a MAP kinase of tobacco. In vitro experiments demonstrated that NopL is phosphorylatyed by SIPK. At least nine distinct spots were observed after two-dimensional gel electrophoresis, indicating that NopL can be hyperphosphorylated by MAP kinases. Senescence symptoms in nodules of beans (Phaseolus vulgaris cv. Tendergreen) were analyzed to determine the symbiotic effector activity of different NopL variants with serine to alanine substitutions at identified and predicted phosphorylation sites (serine-proline motif). NopL variants with six or eight serine to alanine substitutions were partially active, whereas NopL forms with 10 or 12 substituted serine residues were inactive. In conclusion, our findings provide evidence that NopL interacts with MAP kinases and reveals the importance of serine-proline motifs for effector activity during symbiosis.

The Sinorhizobium (Ensifer) fredii HH103 Nodulation Outer Protein NopI Is a Determinant for Efficient Nodulation of Soybean and Cowpea Plants

“…As expected, the cAMP values induced by the NopL-Cya fusion were similar to those obtained in previous studies (18). The fact that the HH103 T3SS genes are expressed in young but not mature soybean nodules and the detection of both NopC and NopL in these young plant organs (18) support the previous idea that NopL acts to prolong the life span of infected cells by suppressing cell death in P. vulgaris nodules (24,25).…”

“…Inactivation of nopL induces the formation of necrotic areas in Phaseolus vulgaris nodules, suggesting a possible role of this putative effector in the suppression of premature senescence (24). Phosphorylation sites of NopL have been identified, and recent results have shown that this protein is localized to the plant nucleus where it forms a complex with salicylic acid-induced protein kinase (SIPK), a tobacco MAPK (25). NopP is also phosphorylated by plant kinases (26), but little is known about the exact function of this effector (27).…”

Know your enemy, embrace your friend: using omics to understand how plants respond differently to pathogenic and mutualistic microorganisms

“…Sinorhizobia sp. also encode the effector NopL that, upon entry into host cells, acts to suppress pathogenesis-related signaling (Bartsev et al, 2004;Ge et al, 2016). In mutualistic fungi, although hundreds of putative effector-like gene sequences have been identified Kohler et al, 2015), to date only three effectors have been functionally characterized.…”

Expression of the Arabidopsis thaliana immune receptor EFR in Medicago truncatula reduces infection by a root pathogenic bacterium, but not nitrogen‐fixing rhizobial symbiosis

“…Sinorhizobium sp. NGR234 translocates multiple type‐3 secreted effectors including NopM, NopL, NopP and NopT to interfere with immune signaling (Dai et al ., ; Ge et al ., ; Skorpil et al ., ; Xin et al ., ). However, the S. meliloti strain 1021 used in our study, Sm 1021, only carries a T4SS gene cluster for translocation of effectors into host cytoplasm, and deletion mutant studies showed no impact of mutating the T4SS on nodulation (Jones et al ., ; Nelson et al ., ).…”