Protist ubiquitin ligase effector PbE3-2 targets cysteine protease RD21A to impede plant immunity.

Clubroot, caused by the soil-borne protist pathogen Plasmodiophora brassicae, is one of the most devastating diseases of Brassica oil and vegetable crops worldwide. Understanding the pathogen infection strategy is crucial for the development of disease control. However, because of its obligate biotrophic nature, the molecular mechanism by which this pathogen promotes infection remains largely unknown. P. brassicae E3 ubiquitin ligase 2 (PbE3-2) is a Really Interesting New Gene (RING)-type E3 ubiquitin ligase in P. brassicae with E3 ligase activity in vitro. Yeast (Saccharomyces cerevisiae) invertase assay and apoplast washing fluid extraction showed that PbE3-2 harbors a functional signal peptide. Overexpression of PbE3-2 in Arabidopsis (Arabidopsis thaliana) resulted in higher susceptibility to P. brassicae and decreases in chitin-triggered reactive oxygen species burst and expression of marker genes in salicylic acid signaling. PbE3-2 interacted with and ubiquitinated host cysteine protease RESPONSIVE TO DEHYDRATION 21A (RD21A) in vitro and in vivo. Mutant plants deficient in RD21A exhibited similar susceptibility and compromised immune responses as in PbE3-2 overexpression plants. We show that PbE3-2, which targets RD21A, is an important virulence factor for P. brassicae. Two other secretory RING-type E3 ubiquitin ligases in P. brassicae performed the same function as PbE3-2 and ubiquitinated RD21A. This study reveals a substantial virulence functional role of protist E3 ubiquitin ligases and demonstrates a mechanism by which protist E3 ubiquitin ligases degrade host immune-associated cysteine proteases to impede host immunity.

A rapid method for detecting and distinguishing metallo-ß-lactamase-and serine carbapenemase-producing Enterobacteriales using MALDI-TOF MS.

Introduction: Carbapenemase-producing Enterobacteriales (CPE) are a major health threat worldwide, and therefore the development of rapid detection methods is needed. Here, we established a method to distinguish metallo-ß-lactamase and serine carbapenemases using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with ethylenediaminetetraacetic acid (EDTA) and phenylboronic acid (PB). Methods: To assess the specificity and sensitivity of the method, 110 carbapenemase-producing and 72 carbapenemase-negative Enterobacteriales isolates were collected, among which 51 strains produced only metallo-ß-lactamase, 55 strains only serine carbapenemases, and four strains both metallo-ß-lactamase and serine carbapenemases. In the proposed MALDI-TOF MS method, imipenem (IPM) and the bacterial strains to be tested were mixed, EDTA and/or PB was added, and the mixture was incubated for 4 h. The carbapenemase type was confirmed by the IPM waveform spectrum before and after incubation. Results: Based on the presence, absence, and recovery of the IPM-cyano-4-hydroxy-cinnamic acid-specific waveform peak near 479 m/z, the detection sensitivity and specificity of the method were 98.2 and 100%, respectively. Discussion: Although CPE detection by MALDI-TOF MS has been studied previously, our method distinguishes between metallo-ß-lactamase and serine carbapenemases, which will be very helpful for the clinical selection of antibiotics.

[Characterization of qnr gene for plasmid-mediated quionlone resistance].

OBJECTIVE: To explore the distribution of qnr gene and broad spectrum p-lactamase (ESBLs) gene in gram-negative bacteria which were isolated from our hospital patients. METHODS: qnr gene in nonrepetitive 129 isolates of Escherichia coli, 10 isolates of Enterobacter cloacac and 29 isolates of K. pneunoniae were detected by polymerase chain reaction (PCR). For qnr gene positive strains, int I, SHV-1, TEM-1, CTX-M, OXA-I , OXA-II , OXA-III, DHA and EBC genes were examined. Plasmid conjugatable test was applied to examine whether qnr gene was located in conjugate plasmid and ERIC-PCR was carried out for DNA homologous analysis. ESBLs detection (according to phenotypic confirmatory test based on National Committee for Clinical Laboratory Standards criteria) and susceptibility test to 16 antibiotics were also performed. RESULTS: qnr gene was found in 6 clinical isolates including 5 strain of E. coli and one strain of E. cloacac, but qnr gene was undetectable in K. pneunoniae isolates. The 6 clinical isolates were suspectible to imipenem but resistance to some other drugs while only 2 isolates of E. coli were susceptible to quinolone. Among the 6 qnr gene-positive strains, all of them belonged to I type integron-positive isolates, 4 isolates of them were TEM-1 producing strains,with only one isolate was OXA-III gene producing strain, and 2 isolates of them were EBC producing strains. Most of them were with 2 ESBLs gene if not more. qnr gene was on transferable plasmids which could be disseminated by clone. CONCLUSION: In Wuhan city, the prevalence of qnr was confirmed. qnr gene were found with some ESBLs gene in the same strains, and qnr gene in suspect strains. The transmission of qnr gene producing strains could be mediated by transferable plasmids or clone, forcing us to make intensive investigation and take effective control measures.