Functional analysis of the type 3 effector nodulation outer protein L (NopL) from Rhizobium sp. NGR234: symbiotic effects, phosphorylation, and interference with mitogen-activated protein kinase signaling.

Pathogenic bacteria use type 3 secretion systems to deliver virulence factors (type 3 effector proteins) directly into eukaryotic host cells. Similarly, type 3 effectors of certain nitrogen-fixing rhizobial strains affect nodule formation in the symbiosis with host legumes. Nodulation outer protein L (NopL) of Rhizobium sp. strain NGR234 is a Rhizobium-specific type 3 effector. Nodulation tests and microscopic analysis showed that distinct necrotic areas were rapidly formed in ineffective nodules of Phaseolus vulgaris (cv. Tendergreen) induced by strain NGRΩnopL (NGR234 mutated in nopL), indicating that NopL antagonized nodule senescence. Further experiments revealed that NopL interfered with mitogen-activated protein kinase (MAPK) signaling in yeast and plant cells (Nicotiana tabacum). Expression of nopL in yeast disrupted the mating pheromone (α-factor) response pathway, whereas nopL expression in N. tabacum suppressed cell death induced either by overexpression of the MAPK gene SIPK (salicylic acid-induced protein kinase) or by SIPK(DD) (mutation in the TXY motif resulting in constitutive MAPK activity). These data indicate that NopL impaired function of MAPK proteins or MAPK substrates. Furthermore, we demonstrate that NopL was multiply phosphorylated either in yeast or N. tabacum cells that expressed nopL. Four phosphorylated serines were confirmed by mass spectrometry. All four phosphorylation sites exhibit a Ser-Pro pattern, a typical motif in MAPK substrates. Taken together, data suggest that NopL mimics a MAPK substrate and that NopL suppresses premature nodule senescence by impairing MAPK signaling in host cells.

Targeted genomic profiling revealed a unique clinical phenotype in intrahepatic cholangiocarcinoma with fibroblast growth factor receptor rearrangement.

Genomic aberrations (GAs) in fibroblast growth factor receptors (FGFRs) are involved in the pathogenesis of intrahepatic cholangiocarcinoma (ICC), and clinical trials have shown efficacy of FGFR inhibitors in treating ICC patients with FGFR GAs such as FGFR2 rearrangement. To clarify the FGFRs GA profile and corresponding clinicopathological features in Chinese patients with ICC, a total of 257 cases were identified. Fourteen cases (5.45%) were positive for FGFR2 rearrangement. Further analysis on the 110 FGFR2 rearrangement negative cases showed that 13 patients present additional FGFRs GAs, including FGFR3 rearrangement (2.73%), and FGFRs mutations. When compared with patients without FGFRs GAs, those with FGFR2 or FGFR3 rearrangement presented more under the age of 58 years, female sex, HBsAb positivity, CD10 expression, and PD-L1 expression. The clinical characteristics between patients with FGFRs mutation and those without FGFRs GAs were similar, with the exception that cases with FGFRs mutation have more hepatolithiasis. We concluded that FGFR rearrangement is associated with unique clinical phenotypes in ICC.