SummaryFive Botrytis cinerea endopolygalacturonase enzymes (BcPGs) were individually expressed in Pichia pastoris, purified to homogeneity and biochemically characterized. While the pH optima of the five enzymes were similar (approximately pH 4.5) the maximum activity of individual enzymes differed significantly. For hydrolysis of polygalacturonic acid (PGA), the V max,app ranged from 10 to 900 U mg )1 , while the K m,app ranged from 0.16 to 0.6 mg ml )1 . Although all BcPGs are true endopolygalacturonases, they apparently have different modes of action. PGA hydrolysis by BcPG1, BcPG2 and BcPG4 leads to the transient accumulation of oligomers with DP < 7, whereas PGA hydrolysis by BcPG3 and BcPG6 leads to the immediate accumulation of monomers and dimers. The necrotizing activity (NA) of all BcPGs was tested separately in tomato, broad bean and Arabidopsis thaliana. They showed different NAs on these plants. BcPG1 and BcPG2 possessed the strongest NA as tissue collapse was observed within 10 min after infiltration of broad bean leaves. The amino acid (aa) D192A substitution in the active site of BcPG2 not only abolished enzyme activity but also the NA, indicating that the NA is dependent on enzyme activity. Furthermore, deletion of the Bcpg2 gene in B. cinerea resulted in a strong reduction in virulence on tomato and broad bean. Primary lesion formation was delayed by approximately 24 h and the lesion expansion rate was reduced by 50-85%. These data indicate that BcPG2 is an important virulence factor for B. cinerea.

Colonization of Arabidopsis thaliana roots by nonpathogenic Pseudomonas fluorescens WCS417r bacteria triggers a jasmonate/ ethylene-dependent induced systemic resistance (ISR) that is effective against a broad range of pathogens. Microarray analysis revealed that the R2R3-MYB-like transcription factor gene MYB72 is specifically activated in the roots upon colonization by WCS417r. Here, we show that T-DNA knockout mutants myb72-1 and myb72-2 are incapable of mounting ISR against the pathogens Pseudomonas syringae pv tomato, Hyaloperonospora parasitica, Alternaria brassicicola, and Botrytis cinerea, indicating that MYB72 is essential to establish broad-spectrum ISR. Overexpression of MYB72 did not result in enhanced resistance against any of the pathogens tested, demonstrating that MYB72 is not sufficient for the expression of ISR. Yeast two-hybrid analysis revealed that MYB72 physically interacts in vitro with the ETHYLENE INSENSITIVE3 (EIN3)-LIKE3 transcription factor EIL3, linking MYB72 function to the ethylene response pathway. However, WCS417r activated MYB72 in ISR-deficient, ethyleneinsensitive ein2-1 plants. Moreover, exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylate induced wild-type levels of resistance in myb72-1, suggesting that MYB72 acts upstream of ethylene in the ISR pathway. Collectively, this study identified the transcriptional regulator MYB72 as a novel ISR signaling component that is required in the roots during early signaling steps of rhizobacteria-mediated ISR.

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An important prerequisite for a conservation programme is a comprehensive description of genetic diversity. The aim of this study was to use anonymous genetic markers to assess the between- and the within-population components of genetic diversity for European pig breeds at the scale of the whole continent using microsatellites. Fifty-eight European pig breeds and lines were analysed including local breeds, national varieties of international breeds and commercial lines. A sample of the Chinese Meishan breed was also included. Eleven additional breeds from a previous project were added for some analyses. Approximately 50 individuals per breed were genotyped for a maximum of 50 microsatellite loci. Substantial within-breed variability was observed, with the average expected heterozygosity and observed number of alleles per locus being 0.56 [range 0.43-0.68] and 4.5 respectively. Genotypic frequencies departed from Hardy-Weinberg expectations (P < 0.01) in 15 European populations, with an excess of homozygotes in 12 of them. The European breeds were on average genetically very distinct, with a Wright F(ST) index value of 0.21. The Neighbour-Joining tree drawn from the Reynolds distances among the breeds showed that the national varieties of major breeds and the commercial lines were mostly clustered around their breeds of reference (Duroc, Hampshire, Landrace, Large White and Piétrain). In contrast, local breeds, with the exception of the Iberian breeds, exhibited a star-like topology. The results are discussed in the light of various forces, which may have driven the recent evolution of European pig breeds. This study has consequences for the interpretation of biodiversity results and will be of importance for future conservation programmes.

The replication/transcription complex of the arterivirus equine arteritis virus (EAV) is associated with paired membranes and/or double-membrane vesicles (DMVs) that are thought to originate from the endoplasmic reticulum. Previously, coexpression of two putative transmembrane nonstructural proteins (nsp2 and nsp3) was found to suffice to induce these remarkable membrane structures, which are typical of arterivirus infection. Here, site-directed mutagenesis was used to investigate the role of nsp3 in more detail. Liberation of the hydrophobic N terminus of nsp3, which is normally achieved by cleavage of the nsp2/3 junction by the nsp2 protease, was nonessential for the formation of DMVs. However, the substitution of each of a cluster of four conserved cysteine residues, residing in a predicted luminal loop of nsp3, completely blocked DMV formation. Some of these mutant nsp3 proteins were also found to be highly cytotoxic, in particular, exerting a dramatic effect on the endoplasmic reticulum. The functionality of an engineered N glycosylation site in the cysteine-containing loop confirmed both its presence in the lumen and the transmembrane nature of nsp3. This mutant displayed an interesting intermediate phenotype in terms of DMV formation, with paired and curved membranes being formed, but DMV formation apparently being impaired. The effect of nsp3 mutations on replicase polyprotein processing was investigated, and several mutations were found to influence processing of the region downstream of nsp3 by the nsp4 main protease. When tested in an EAV reverse genetics system, none of the nsp3 mutations was tolerated, again underlining the crucial role of the protein in the arterivirus life cycle.The replication or replication/transcription complexes (RTCs) of a wide variety of eukaryotic positive-strand RNA viruses have been found to be associated with (modified) intracellular membranes (for recent reviews, see references 1, 2, 22, 24, 25, and 31). Membrane association of the RTC is thought to be important for creating a suitable (micro)environment for viral RNA synthesis and may also aid in preventing the activation of host defense mechanisms that can be triggered by double-stranded RNA replication intermediates. For several virus groups, replicase subunits have been identified that are involved in targeting the RTC to membrane compartments and/or modifying these membranes, often resulting in vesiculation or the formation of invaginations. Frequently, parts of these nonstructural proteins, which often contain multiple hydrophobic segments, are known or thought to be embedded in the membrane. All major groups of mammalian positive-strand RNA viruses produce their replicative machinery from replicase polyproteins containing both these hydrophobic subunits and the enzymes directly involved in RNA synthesis. Consequently, both the correct proteolytic processing and the membrane association of replicase subunits are important and probably highly coordinated events during the initial stages of the viral life cycle.Equi...

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CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, Campylobacter jejuni secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of Streptococcus pyogenes (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications.