Three proline rotamases involved in calcium homeostasis play differential roles in stress tolerance,virulence and calcineurin regulation of Beauveria bassiana

FK506‐sensitive proline rotamases (FPRs), also known as FK506‐binding proteins (FKBPs), can mediate immunosuppressive drug resistance in budding yeast but their physiological roles in filamentous fungi remain opaque. Here, we report that three FPRs (cytosolic/nuclear 12.15‐kD Fpr1, membrane‐associated 14.78‐kD Fpr2 and nuclear 50.43‐kD Fpr3) are all equally essential for cellular Ca²⁺ homeostasis and contribute significantly to calcineurin activity at different levels in the insect‐pathogenic fungus Beauveria bassiana although the deletion of fpr1 alone conferred resistance to FK506. Radial growth, conidiation, conidial viability and virulence were less compromised in the absence of fpr1 or fpr2 than in the absence of fpr3, which abolished almost all growth on scant media and reduced growth moderately on rich media. The Δfpr3 mutant was more sensitive to Na⁺, K⁺, Mn²⁺, Ca²⁺, Cu²⁺, metal chelate, heat shock and UVB irradiation than was Δfpr2 while both mutants were equally sensitive to Zn²⁺, Mg²⁺, Fe²⁺, H₂O₂ and cell wall‐perturbing agents. In contrast, the Δfpr1 mutant was less sensitive to fewer stress cues. Most of 32 examined genes involved in DNA damage repair, Na⁺/K⁺ detoxification or osmotolerance and Ca²⁺ homeostasis were downregulated sharply in Δfpr2 and Δfpr3 but rarely so affected in Δfpr1, coinciding well with their phenotypic changes. These findings uncover important, but differential, roles of three FPRs in the fungal adaptation to insect host and environment and provide novel insight into their essential roles in calcium signalling pathway.     

PagR mediates the precise regulation of Salmonella pathogenicity island 2 gene expression in response to magnesium and phosphate signals in Salmonella Typhimurium

To establish systemic infections, Salmonella enterica serovar Typhimurium (S. Typhimurium) requires Salmonella pathogenicity island 2 (SPI‐2) to survive and replicate within macrophages. High expression of many SPI‐2 genes during the entire intracellular growth period within macrophages is essential, as it contributes to the formation of Salmonella‐containing vacuole and bacterial replication. However, the regulatory mechanisms underlying the sustained induction of SPI‐2 within macrophages are not fully understood. Here, we revealed a time‐dependent regulation of SPI‐2 expression mediated by a novel regulator PagR (STM2345) in response to the low Mg²⁺ and low phosphate (P_i) signals, which ensured the high induction of SPI‐2 during the entire intramacrophage growth period. Deletion of pagR results in reduced bacterial replication in macrophages and attenuation of systemic virulence in mice. The effects of pagR on virulence are dependent on upregulating the expression of slyA, a regulator of SPI‐2. At the early (0–4 hr) and later (after 4 hr) stage post‐infection of macrophages, pagR is induced by the low P_i via PhoB/R two‐component systems and low Mg²⁺ via PhoP/Q systems, respectively. Collectively, our findings revealed that the PagR‐mediated regulatory mechanism contributes to the precise and sustained activation of SPI‐2 genes within macrophages, which is essential for S. Typhimurium systemic virulence.     

Meningococcal disease: A paradigm of type‐IV pilus dependent pathogenesis

Neisseria meningitidis (meningococcus) is a Gram‐negative bacterium responsible for two devastating forms of invasive diseases: purpura fulminans and meningitis. Interaction with both peripheral and cerebral microvascular endothelial cells is at the heart of meningococcal pathogenesis. During the last two decades, an essential role for meningococcal type IV pili in vascular colonisation and disease progression has been unravelled. This review summarises 20 years of research on meningococcal type IV pilus‐dependent virulence mechanisms, up to the identification of promising anti‐virulence compounds that target type IV pili.     

Two Phytophthora parasitica cysteine protease genes,PpCys44 and PpCys45, trigger cell death in various Nicotiana spp. and act as virulence factors

Proteases secreted by pathogens have been shown to be important virulence factors modifying plant immunity, and cysteine proteases have been demonstrated to participate in different pathosystems. However, the virulence functions of the cysteine proteases secreted by Phytophthora parasitica are poorly understood. Using a publicly available genome database, we identified 80 cysteine proteases in P. parasitica, 21 of which were shown to be secreted. Most of the secreted cysteine proteases are conserved among different P. parasitica strains and are induced during infection. The secreted cysteine protease proteins PpCys44/45 (proteases with identical protein sequences) and PpCys69 triggered cell death on the leaves of different Nicotiana spp. A truncated mutant of PpCys44/45 lacking a signal peptide failed to trigger cell death, suggesting that PpCys44/45 functions in the apoplastic space. Analysis of three catalytic site mutants showed that the enzyme activity of PpCys44/45 is required for its ability to trigger cell death. A virus-induced gene silencing assay showed that PpCys44/45 does not induce cell death on NPK1 (Nicotiana Protein Kinase 1)-silenced Nicotiana benthamiana plants, indicating that the cell death phenotype triggered by PpCys44/45 is dependent on NPK1. PpCys44- and PpCys45-deficient double mutants showed decreased virulence, suggesting that PpCys44 and PpCys45 positively promote pathogen virulence during infection. PpCys44 and PpCys45 are important virulence factors of P. parasitica and trigger NPK1-dependent cell death in various Nicotiana spp.     

Functional analyses of small secreted cysteine-rich proteins identified candidate effectors in Verticillium dahliae

Secreted small cysteine-rich proteins (SCPs) play a critical role in modulating host immunity in plant–pathogen interactions. Bioinformatic analyses showed that the fungal pathogen Verticillium dahliae encodes more than 100 VdSCPs, but their roles in host–pathogen interactions have not been fully characterized. Transient expression of 123 VdSCP-encoding genes in Nicotiana benthamiana identified three candidate genes involved in host–pathogen interactions. The expression of these three proteins, VdSCP27, VdSCP113, and VdSCP126, in N. benthamiana resulted in cell death accompanied by a reactive oxygen species burst, callose deposition, and induction of defence genes. The three VdSCPs mainly localized to the periphery of the cell. BAK1 and SOBIR1 (associated with receptor-like protein) were required for the immunity triggered by these three VdSCPs in N. benthamiana. Site-directed mutagenesis showed that cysteine residues that form disulphide bonds are essential for the functioning of VdSCP126, but not VdSCP27 and VdSCP113. VdSCP27, VdSCP113, and VdSCP126 individually are not essential for V. dahliae infection of N. benthamiana and Gossypium hirsutum, although there was a significant reduction of virulence on N. benthamiana and G. hirsutum when inoculated with the VdSCP27/VdSCP126 double deletion strain. These results illustrate that the SCPs play a critical role in the V. dahliae–plant interaction via an intrinsic virulence function and suppress immunity following infection.     

Plasmid composition and the chpG gene determine the virulence level of Clavibacter capsici natural isolates in pepper

The gram-positive bacterial species Clavibacter capsici causes necrosis and canker in pepper plants. Genomic and functional analyses of C. capsici type strain PF008 have shown that multiple virulence genes exist in its two plasmids. We aimed to identify the key determinants that control the virulence of C. capsici. Pepper leaves inoculated with 54 natural isolates exhibited significant variation in the necrosis. Six isolates showed very low virulence, but their population titres in plants were not significantly different from those of the highly virulent isolates. All six isolates lacked the pCM1_Cc plasmid that carries chpG, which has been shown to be required for virulence and encodes a putative serine protease, but two of them, isolates 1,106 and 1,207, had the intact chpG elsewhere in the genome. Genomic analysis of these two isolates revealed that chpG was located in the pCM2_Cc plasmid, and two highly homologous regions were present next to the chpG locus. The chpG expression in isolate 1,106 was not induced in plants. Introduction of chpG of the PF008 strain into the six low-virulence isolates restored their virulence to that of PF008. Our findings indicate that there are at least three different variant groups of C. capsici and that the plasmid composition and the chpG gene are critical for determining the virulence level. Moreover, our findings also indicate that the virulence level of C. capsici does not directly correlate with bacterial titres in plants.     

Exposure to noise pollution across North American passerines supports the noise filter hypothesis

The noise filter hypothesis predicts that species using higher sound frequencies should be more tolerant of noise pollution, because anthropogenic noise is more intense at low frequencies. Recent work analysed continental‐scale data on anthropogenic noise across the USA and found that passerine species inhabiting more noise‐polluted areas do not have higher peak song frequency but have more complex songs. However, this metric of song complexity is of ambiguous interpretation, because it can indicate either diverse syllables or a larger frequency bandwidth. In the latter case, the finding would support the noise filter hypothesis, because larger frequency bandwidths mean that more sound energy spreads to frequencies that are less masked by anthropogenic noise. We reanalysed how passerine song predicts exposure to noise using a more thorough dataset of acoustic song measurements, and showed that it is large frequency bandwidths, rather than diverse syllables, that predict the exposure of species to noise pollution. Given that larger bandwidths often encompass higher maximum frequencies, which are less masked by anthropogenic noise, our result suggests that tolerance to noise pollution might depend mostly on having the high‐frequency parts of song little masked by noise, thus preventing acoustic communication from going entirely unnoticed at long distances.