The CfMK1 Gene Regulates Reproduction, Appressorium Formation, and Pathogenesis in a Pear Anthracnose-Causing Fungus

Colletotrichum fructicola, the causal agent of pear anthracnose, causes significant annual economic losses. Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways that play a crucial role in mediating cellular responses to environmental and host signals in plant pathogenic fungi. In this study, we identified an ortholog of the FUS3/KSS1-related MAPK gene, CfMK1, and characterized its function in C. fructicola. The Cfmk1 deletion mutants exhibited poorly developed aerial hyphae, autolysis, no conidial mass or perithecia on solid plates. However, the conidiation of the Cfmk1 mutant in PDB liquid medium was normal compared with that of the wild type (WT). Conidia of the Cfmk1 mutant exhibited a reduced germination rate on glass slides or plant surfaces. The Cfmk1 deletion mutants were unable to form appressoria and lost the capacity to penetrate plant epidermal cells. The ability of the Cfmk1 mutants to infect pear leaves and fruit was severely reduced. Moreover, RNA sequencing (RNA-seq) analysis of the WT and Cfmk1 mutant was performed, and the results revealed 1886 upregulated and 1554 downregulated differentially expressed genes (DEGs) in the mutant. The DEGs were significantly enriched in cell wall and pathogenesis terms, which was consistent with the defects of the Cfmk1 mutant in cell wall integrity and plant infection. Overall, our data demonstrate that CfMK1 plays critical roles in the regulation of aerial hyphal growth, asexual and sexual reproduction, autolysis, appressorium formation, and pathogenicity.

Genome-Wide Identification of bZIP Transcription Factor Genes and Functional Analyses of Two Members in Cytospora chrysosperma

The basic leucine zipper (bZIP) transcription factor (TF) family, one of the largest and the most diverse TF families, is widely distributed across the eukaryotes. It has been described that the bZIP TFs play diverse roles in development, nutrient utilization, and various stress responses in fungi. However, little is known of the bZIP members in Cytospora chrysosperma, a notorious plant pathogenic fungus, which causes canker disease on over 80 woody plant species. In this study, 26 bZIP genes were systematically identified in the genome of C. chrysosperma, and two of them (named CcbZIP05 and CcbZIP23) significantly down-regulated in CcPmk1 deletion mutant (a pathogenicity-related mitogen-activated protein kinase) were selected for further analysis. Deletion of CcbZIP05 or CcbZIP23 displayed a dramatic reduction in fungal growth but showed increased hypha branching and resistance to cell wall inhibitors and abiotic stresses. The CcbZIP05 deletion mutants but not CcbZIP23 deletion mutants were more sensitive to the hydrogen peroxide compared to the wild-type and complemented strains. Additionally, the CcbZIP23 deletion mutants produced few pycnidia but more pigment. Remarkably, both CcbZIP05 and CcbZIP23 deletion mutants were significantly reduced in fungal virulence. Further analysis showed that CcbZIP05 and CcbZIP23 could regulate the expression of putative effector genes and chitin synthesis-related genes. Taken together, our results suggest that CcbZIP05 and CcbZIP23 play important roles in fungal growth, abiotic stresses response, and pathogenicity, which will provide comprehensive information on the CcbZIP genes and lay the foundation for further research on the bZIP members in C. chrysosperma.

Cold Shock Proteins Promote Nisin Tolerance in Listeria monocytogenes Through Modulation of Cell Envelope Modification Responses

Listeria monocytogenes continues to be a food safety challenge owing to its stress tolerance and virulence traits. Several listeriosis outbreaks have been linked to the consumption of contaminated ready-to-eat food products. Numerous interventions, including nisin application, are presently employed to mitigate against L. monocytogenes risk in food products. In response, L. monocytogenes deploys several defense mechanisms, reducing nisin efficacy, that are not yet fully understood. Cold shock proteins (Csps) are small, highly conserved nucleic acid-binding proteins involved in several gene regulatory processes to mediate various stress responses in bacteria. L. monocytogenes possesses three csp gene paralogs; cspA, cspB, and cspD. Using a panel of single, double, and triple csp gene deletion mutants, the role of Csps in L. monocytogenes nisin tolerance was examined, demonstrating their importance in nisin stress responses of this bacterium. Without csp genes, a L. monocytogenes ΔcspABD mutant displayed severely compromised growth under nisin stress. Characterizing single (ΔcspA, ΔcspB, and ΔcspD) and double (ΔcspBD, ΔcspAD, and ΔcspAB) csp gene deletion mutants revealed a hierarchy (cspD > cspB > cspA) of importance in csp gene contributions toward the L. monocytogenes nisin tolerance phenotype. Individual eliminations of either cspA or cspB improved the nisin stress tolerance phenotype, suggesting that their expression has a curbing effect on the expression of nisin resistance functions through CspD. Gene expression analysis revealed that Csp deficiency altered the expression of DltA, MprF, and penicillin-binding protein-encoding genes. Furthermore, the ΔcspABD mutation induced an overall more electronegative cell surface, enhancing sensitivity to nisin and other cationic antimicrobials as well as the quaternary ammonium compound disinfectant benzalkonium chloride. These observations demonstrate that the molecular functions of Csps regulate systems important for enabling the constitution and maintenance of an optimal composed cell envelope that protects against cell-envelope-targeting stressors, including nisin. Overall, our data show an important contribution of Csps for L. monocytogenes stress protection in food environments where antimicrobial peptides are used. Such knowledge can be harnessed in the development of better L. monocytogenes control strategies. Furthermore, the potential that Csps have in inducing cross-protection must be considered when combining hurdle techniques or using them in a series.

Biofilm Interaction Mapping and Analysis (BIMA) of Interspecific Interactions in Pseudomonas Co-culture Biofilms

Pseudomonas species are ubiquitous in nature and include numerous medically, agriculturally and technologically beneficial strains of which the interspecific interactions are of great interest for biotechnologies. Specifically, co-cultures containing Pseudomonas stutzeri have been used for bioremediation, biocontrol, aquaculture management and wastewater denitrification. Furthermore, the use of P. stutzeri biofilms, in combination with consortia-based approaches, may offer advantages for these processes. Understanding the interspecific interaction within biofilm co-cultures or consortia provides a means for improvement of current technologies. However, the investigation of biofilm-based consortia has been limited. We present an adaptable and scalable method for the analysis of macroscopic interactions (colony morphology, inhibition, and invasion) between colony-forming bacterial strains using an automated printing method followed by analysis of the genes and metabolites involved in the interactions. Using Biofilm Interaction Mapping and Analysis (BIMA), these interactions were investigated between P. stutzeri strain RCH2, a denitrifier isolated from chromium (VI) contaminated soil, and 13 other species of pseudomonas isolated from non-contaminated soil. One interaction partner, Pseudomonas fluorescens N1B4 was selected for mutant fitness profiling of a DNA-barcoded mutant library; with this approach four genes of importance were identified and the effects on interactions were evaluated with deletion mutants and mass spectrometry based metabolomics.

Behavioural phenotyping of C. elegans on UV-killed E. coli mutants v1

Protocol for screening candidate behaviour-modifying E. coli BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their effects on Caenorhabditis elegans behaviour when killed by ultraviolet (UV) light

Coordinated Regulation of Protoperithecium Development by MAP Kinases MAK-1 and MAK-2 in Neurospora crassa

Mitogen-activated protein (MAP) kinase pathways function as signaling hubs that are integral for many essential cellular processes, including sexual development. The molecular mechanisms and cross-talk between PR and CWI MAP kinase pathways have been extensively studied during asexual development. However, if these can be extended to sexual development remains elusive. By analyzing genome-wide transcriptional responses to deletion of each of two MAP kinase coding genes mak-2 (PR-MAP kinase pathway) and mak-1 (CWI-MAP kinase pathway) in Neurospora crassa during protoperithecium formation, 430 genes co-regulated by the MAK-1 and MAK-2 proteins were found, functionally enriched at integral components of membrane and oxidoreductase. These genes include 13 functionally known genes participating in sexual development (app, poi-2, stk-17, fsd-1, vsd-8, and NCU03863) and melanin synthesis (per-1, pkh-1, pkh-2, mld-1, scy-1, trn-2, and trn-1), as well as a set of functionally unknown genes. Phenotypic analysis of deletion mutants for the functionally unknown genes revealed that 12 genes were essential for female fertility. Among them, single-gene deletion mutants for NCU07743 (named as pfd-1), NCU02250 (oli), and NCU05948 (named as pfd-2) displayed similar protoperithecium development defects as the Δmak-1 and Δmak-2 mutants, failing to form protoperithecium. Western blotting analysis showed that both phosphorylated and total MAK-1 proteins were virtually abolished in the Δnrc-1, Δmek-2, and Δmak-2 mutants, suggesting that the posttranscriptional regulation of MAK-1 is dependent on the PR-MAP kinase pathway during the protoperithecium development. Taken together, this study revealed the regulatory roles and cross-talk between PR and CWI-MAP kinase pathways during protoperithecium development.

Predicted transmembrane proteins with homology to Mef(A) are not responsible for complementing mef(A) deletion in the mef(A)–msr(D) macrolide efflux system in Streptococcus pneumoniae

Objectives In streptococci, the type M resistance to macrolides is due to the mef(A)–msr(D) efflux transport system of the ATP-Binding cassette (ABC) superfamily, where it is proposed that mef(A) codes for the transmembrane channel and msr(D) for the two ATP-binding domains. Phage ϕ1207.3 of Streptococcus pyogenes, carrying the mef(A)–msr(D) gene pair, is able to transfer the macrolide efflux phenotype to Streptococcus pneumoniae. Deletion of mef(A) in pneumococcal ϕ1207.3-carrying strains did not affect erythromycin efflux. In order to identify candidate genes likely involved in complementation of mef(A) deletion, the Mef(A) amino acid sequence was used as probe for database searching. Results In silico analysis identified 3 putative candidates in the S. pneumoniae R6 genome, namely spr0971, spr1023 and spr1932. Isogenic deletion mutants of each candidate gene were constructed and used in erythromycin sensitivity assays to investigate their contribution to mef(A) complementation. Since no change in erythromycin sensitivity was observed compared to the parental strain, we produced double and triple mutants to assess the potential synergic activity of the selected genes. Also these mutants did not complement the mef(A) function.