Susceptibility Testing of Environmental and Clinical Aspergillus sydowii Demonstrates Potent Activity of Various Antifungals.

The genus Aspergillus consists of a vast number of medically and environmentally relevant species. Aspergillus species classified in series Versicolores are ubiquitous in the environment and include the opportunistic pathogen Aspergillus sydowii, which is associated with onychomycosis and superficial skin infections. Despite frequent clinical reports of A. sydowii and related series Versicolores species, antifungal susceptibility data are scarce, hampering optimal treatment choices and subsequent patient outcomes. Here, we employed antifungal susceptibility testing (AFST) based on microbroth dilution on a set of 155 series Versicolores strains using the common antifungals amphotericin B, itraconazole, voriconazole, posaconazole, isavuconazole and micafungin with the addition of luliconazole and olorofim. All strains were identified using partial calmodulin gene sequencing, with 145 being A. sydowii, seven A. creber and three A. versicolor, using the latest taxonomic insights. Overall, tested antifungals were potent against the entire strain collection. In comparison to A. fumigatus, azole and amphotericin B MICs were slightly elevated for some strains. AFST with luliconazole and olorofim, here reported for the first time, displayed the highest in vitro activity, making these antifungals interesting alternative drugs but clinical studies are warranted for future therapeutic use.

Genome-wide identification and expression analysis of calmodulin and calmodulin-like genes in passion fruit (Passiflora edulis) and their involvement in flower and fruit development.

BACKGROUND: The calmodulin (CaM) and calmodulin-like (CML) proteins play regulatory roles in plant growth and development, responses to biotic and abiotic stresses, and other biological processes. As a popular fruit and ornamental crop, it is important to explore the regulatory mechanism of flower and fruit development of passion fruit. RESULTS: In this study, 32 PeCaM/PeCML genes were identified from passion fruit genome and were divided into 9 groups based on phylogenetic analysis. The structural analysis, including conserved motifs, gene structure and homologous modeling, illustrates that the PeCaM/PeCML in the same subgroup have relative conserved structural features. Collinearity analysis suggested that the expansion of the CaM/CML gene family likely took place mainly by segmental duplication, and the whole genome replication events were closely related with the rapid expansion of the gene group. PeCaM/PeCMLs were potentially required for different floral tissues development. Significantly, PeCML26 had extremely high expression levels during ovule and fruit development compared with other PeCML genes, suggesting that PeCML26 had potential functions involved in the development of passion fruit flowers and fruits. The co-presence of various cis-elements associated with growth and development, hormone responsiveness, and stress responsiveness in the promoter regions of these PeCaM/PeCMLs might contribute to their diverse regulatory roles. Furthermore, PeCaM/PeCMLs were also induced by various abiotic stresses. This work provides a comprehensive understanding of the CaM/CML gene family and valuable clues for future studies on the function and evolution of CaM/CML genes in passion fruit. CONCLUSION: A total of 32 PeCaM/PeCML genes were divided into 9 groups. The PeCaM/PeCML genes showed differential expression patterns in floral tissues at different development stages. It is worth noting that PeCML26, which is highly homologous to AtCaM2, not only interacts with multiple BBR-BPC TFs, but also has high expression levels during ovule and fruit development, suggesting that PeCML26 had potential functions involved in the development of passion fruit flowers and fruits. This research lays the foundation for future investigations and validation of the potential function of PeCaM/PeCML genes in the growth and development of passion fruit.

Positive roles of the Ca2+ sensors GbCML45 and GbCML50 in improving cotton Verticillium wilt resistance.

As a universal second messenger, cytosolic calcium (Ca2+) functions in multifaceted intracellular processes, including growth, development and responses to biotic/abiotic stresses in plant. The plant-specific Ca2+ sensors, calmodulin and calmodulin-like (CML) proteins, function as members of the second-messenger system to transfer Ca2+ signal into downstream responses. However, the functions of CMLs in the responses of cotton (Gossypium spp.) after Verticillium dahliae infection, which causes the serious vascular disease Verticillium wilt, remain elusive. Here, we discovered that the expression level of GbCML45 was promoted after V. dahliae infection in roots of cotton, suggesting its potential role in Verticillium wilt resistance. We found that knockdown of GbCML45 in cotton plants decreased resistance while overexpression of GbCML45 in Arabidopsis thaliana plants enhanced resistance to V. dahliae infection. Furthermore, there was physiological interaction between GbCML45 and its close homologue GbCML50 by using yeast two-hybrid and bimolecular fluorescence assays, and both proteins enhanced cotton resistance to V. dahliae infection in a Ca2+-dependent way in a knockdown study. Detailed investigations indicated that several defence-related pathways, including salicylic acid, ethylene, reactive oxygen species and nitric oxide signalling pathways, as well as accumulations of lignin and callose, are responsible for GbCML45- and GbCML50-modulated V. dahliae resistance in cotton. These results collectively indicated that GbCML45 and GbCML50 act as positive regulators to improve cotton Verticillium wilt resistance, providing potential targets for exploitation of improved Verticillium wilt-tolerant cotton cultivars by genetic engineering and molecular breeding.

Development of an miRFP680-Based Fluorescent Calcium Ion Biosensor Using End-Optimized Transposons.

The development of new or improved single fluorescent protein (FP)-based biosensors (SFPBs), particularly those with excitation and emission at near-infrared wavelengths, is important for the continued advancement of biological imaging applications. In an effort to accelerate the development of new SFPBs, we report modified transposons for the transposase-based creation of libraries of FPs randomly inserted into analyte binding domains, or vice versa. These modified transposons feature ends that are optimized to minimize the length of the linkers that connect the FP to the analyte binding domain. We rationalized that shorter linkers between the domains should result in more effective allosteric coupling between the analyte binding-dependent conformational change in the binding domain and the fluorescence modulation of the chromophore of the FP domain. As a proof of concept, we employed end-modified Mu transposons for the discovery of SFPB prototypes based on the insertion of two circularly permuted red FPs (mApple and FusionRed) into binding proteins for l-lactate and spermidine. Using an analogous approach, we discovered calcium ion (Ca2+)-specific SFPBs by random insertion of calmodulin (CaM)-RS20 into miRFP680, a particularly bright near-infrared (NIR) FP based on a biliverdin (BV)-binding fluorescent protein. Starting from an miRFP680-based Ca2+ biosensor prototype, we performed extensive directed evolution, including under BV-deficient conditions, to create highly optimized biosensors designated the NIR-GECO3 series. We have extensively characterized the NIR-GECO3 series and explored their utility for biological Ca2+ imaging. The methods described in this work will serve to accelerate SFPB development and open avenues for further exploration and optimization of SFPBs across a spectrum of biological applications.

Functional validation of AaCaM3 response to high temperature stress in Amorphophallus albus.

Amorphophallus is a perennial monocotyledonous herbaceous plant native to the southwestern region of China, widely used in various fields such as food processing, biomedicine and chemical agriculture. However, Amorphophallus is a typical thermolabile plant, and the continuous high temperature in summer have seriously affected the growth, development and economic yield of Amorphophallus in recent years. Calmodulin (CaM), a Ca2+ sensor ubiquitous in eukaryotes, is the most important multifunctional receptor protein in plant cells, which affects plant stress resistance by participating in the activities of a variety of signaling molecules. In this study, the key gene AaCaM3 for the Ca2+-CaM regulatory pathway was obtained from A. albus, the sequence analysis confirmed that it is a typical calmodulin. The qRT-PCR results demonstrated that with the passage of heat treatment time, the expression of AaCaM3 was significantly upregulated in A. albus leaves. Subcellular localization analysis revealed that AaCaM3 localized on the cytoplasm and nucleus. Meanwhile, heterologous transformation experiments have shown that AaCaM3 can significantly improve the heat tolerance of Arabidopsis under heat stress. The promoter region of AaCaM3 was sequenced 1,338 bp by FPNI-PCR and GUS staining assay showed that the promoter of AaCaM3 was a high-temperature inducible promoter. Yeast one-hybrid analysis and Luciferase activity reporting system analysis showed that the AaCaM3 promoter may interact with AaHSFA1, AaHSFA2c, AaHSP70, AaDREB2a and AaDREB2b. In conclusion, this study provides new ideas for further improving the signal transduction network of high-temperature stress in Amorphophallus.

Gossypium hirsutum calmodulin-like protein (CML 11) interaction with geminivirus encoded protein using bioinformatics and molecular techniques.

Plant viruses are the most abundant destructive agents that exist in every ecosystem, causing severe diseases in multiple crops worldwide. Currently, a major gap is present in computational biology determining plant viruses interaction with its host. We lay out a strategy to extract virus-host protein interactions using various protein binding and interface methods for Geminiviridae, a second largest virus family. Using this approach, transcriptional activator protein (TrAP/C2) encoded by Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan virus (CLCuMV) showed strong binding affinity with calmodulin-like (CML) protein of Gossypium hirsutum (Gh-CML11). Higher negative value for the change in Gibbs free energy between TrAP and Gh-CML11 indicated strong binding affinity. Consensus from gene ontology database and in-silico nuclear localization signal (NLS) tools identified subcellular localization of TrAP in the nucleus associated with Gh-CML11 for virus infection. Data based on interaction prediction and docking methods present evidences that full length and truncated C2 strongly binds with Gh-CML11. This computational data was further validated with molecular results collected from yeast two-hybrid, bimolecular fluorescence complementation system and pull down assay. In this work, we also show the outcomes of full length and truncated TrAP on plant machinery. This is a first extensive report to delineate a role of CML protein from cotton with begomoviruses encoded transcription activator protein.

Calmodulin-like 5 promotes PEDV replication by regulating late-endosome synthesis and innate immune response.

The infection caused by porcine epidemic diarrhea virus (PEDV) is associated with high mortality in piglets worldwide. Host factors involved in the efficient replication of PEDV, however, remain largely unknown. Our recent proteomic study in the virus-host interaction network revealed a significant increase in the accumulation of CALML5 (EF-hand protein calmodulin-like 5) following PEDV infection. A further study unveiled a biphasic increase of CALML5 in 2 and 12 â€‹h after viral infection. Similar trends were observed in the intestines of piglets in the early and late stages of the PEDV challenge. Moreover, CALML5 depletion reduced PEDV mRNA and protein levels, leading to a one-order-of-magnitude decrease in virus titer. At the early stage of PEDV infection, CALML5 affected the endosomal trafficking pathway by regulating the expression of endosomal sorting complex related cellular proteins. CALML5 depletion also suppressed IFN-ß and IL-6 production in the PEDV-infected cells, thereby indicating its involvement in negatively regulating the innate immune response. Our study reveals the biological function of CALML5 in the virology field and offers new insights into the PEDV-host cell interaction.

mRNA Display Pipeline for Protein Biosensor Construction.

Despite the significant potential of protein biosensors, their construction remains a trial-and-error process. The most obvious approach for addressing this is to utilize modular biosensor architectures where specificity-conferring modalities can be readily generated to recognize new targets. Toward this goal, we established a workflow that uses mRNA display-based selection of hyper-stable monobody domains for the target of choice or ribosome display to select equally stable DARPins. These binders were integrated into a two-component allosteric biosensor architecture based on a calmodulin-reporter chimera. This workflow was tested by developing biosensors for liver toxicity markers such as cytosolic aspartate aminotransferase, mitochondrial aspartate aminotransferase, and alanine aminotransferase 1. We demonstrate that our pipeline consistently produced >103 unique binders for each target within a week. Our analysis revealed that the affinity of the binders for their targets was not a direct predictor of the binder's performance in a biosensor context. The interactions between the binding domains and the reporter module affect the biosensor activity and the dynamic range. We conclude that following binding domain selection, the multiplexed biosensor assembly and prototyping appear to be the most promising approach for identifying biosensors with the desired properties.

High-throughput functional mapping of variants in an arrhythmia gene, KCNE1, reveals novel biology.

BACKGROUND: KCNE1 encodes a 129-residue cardiac potassium channel (IKs) subunit. KCNE1 variants are associated with long QT syndrome and atrial fibrillation. However, most variants have insufficient evidence of clinical consequences and thus limited clinical utility. METHODS: In this study, we leveraged the power of variant effect mapping, which couples saturation mutagenesis with high-throughput sequencing, to ascertain the function of thousands of protein-coding KCNE1 variants. RESULTS: We comprehensively assayed KCNE1 variant cell surface expression (2554/2709 possible single-amino-acid variants) and function (2534 variants). Our study identified 470 loss- or partial loss-of-surface expression and 574 loss- or partial loss-of-function variants. Of the 574 loss- or partial loss-of-function variants, 152 (26.5%) had reduced cell surface expression, indicating that most functionally deleterious variants affect channel gating. Nonsense variants at residues 56-104 generally had WT-like trafficking scores but decreased functional scores, indicating that the latter half of the protein is dispensable for protein trafficking but essential for channel function. 22 of the 30 KCNE1 residues (73%) highly intolerant of variation (with > 70% loss-of-function variants) were in predicted close contact with binding partners KCNQ1 or calmodulin. Our functional assay data were consistent with gold standard electrophysiological data (ρ = - 0.64), population and patient cohorts (32/38 presumed benign or pathogenic variants with consistent scores), and computational predictors (ρ = - 0.62). Our data provide moderate-strength evidence for the American College of Medical Genetics/Association of Molecular Pathology functional criteria for benign and pathogenic variants. CONCLUSIONS: Comprehensive variant effect maps of KCNE1 can both provide insight into I Ks channel biology and help reclassify variants of uncertain significance.

EP4-induced mitochondrial localization and cell migration mediated by CALML6 in human oral squamous cell carcinoma.

Lymph node metastasis, primarily caused by the migration of oral squamous cell carcinoma (OSCC) cells, stands as a crucial prognostic marker. We have previously demonstrated that EP4, a subtype of the prostaglandin E2 (PGE2) receptor, orchestrates OSCC cell migration via Ca2+ signaling. The exact mechanisms by which EP4 influences cell migration through Ca2+ signaling, however, is unclear. Our study aims to clarify how EP4 controls OSCC cell migration through this pathway. We find that activating EP4 with an agonist (ONO-AE1-473) increased intracellular Ca2+ levels and the migration of human oral cancer cells (HSC-3), but not human gingival fibroblasts (HGnF). Further RNA sequencing linked EP4 to calmodulin-like protein 6 (CALML6), whose role remains undefined in OSCC. Through protein-protein interaction network analysis, a strong connection is identified between CALML6 and calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), with EP4 activation also boosting mitochondrial function. Overexpressing EP4 in HSC-3 cells increases experimental lung metastasis in mice, whereas inhibiting CaMKK2 with STO-609 markedly lowers these metastases. This positions CaMKK2 as a potential new target for treating OSCC metastasis. Our findings highlight CALML6 as a pivotal regulator in EP4-driven mitochondrial respiration, affecting cell migration and metastasis via the CaMKK2 pathway.

Calmodulin 2 expression is associated with poor prognosis in breast cancer.

BACKGROUND: Calmodulin 2 (CALM2) belongs to the highly conserved calcium-binding protein family, implicated in the pathogenesis of various malignant tumors. However, its involvement in breast cancer (BRCA) remains unclear. This study aimed to examine CALM2 expression in BRCA and its associations with prognosis, clinicopathological features, protein-protein interactions, and immune cell infiltration. MATERIALS AND METHODS: Online bioinformatics tools were employed to assess CALM2 expression and its clinical relevance in BRCA. Western blotting and immunohistochemistry were utilized to evaluate CALM2 expression in BRCA cell lines and tissues. Logistic regression was applied to analyze the relationship between CALM2 expression levels and clinicopathological parameters. Transwell assay was performed to validate the role of CALM2 in BRCA migration and invasion. RESULTS: CALM2 expression was significantly elevated in BRCA, with increased levels predicting poor overall survival (OS) and disease-free survival (DFS). Moreover, high CALM2 expression correlated with poorer DFS specifically in triple-negative breast cancer (TNBC). CALM2 expression in BRCA showed significant associations with lymph node metastasis, TP53 mutation status, and menopause status. Silencing CALM2 in BRCA cells demonstrated inhibition of cell migration and invasion in vitro. CONCLUSIONS: CALM2 is overexpressed in BRCA and its upregulation is significantly correlated with poor patient prognosis. Elevated CALM2 expression holds promise as a potential molecular marker for predicting poor survival and as a therapeutic target in BRCA.

A calmodulin-like protein PvCML9 negatively regulates salt tolerance.

Calmodulin-like proteins (CMLs) are unique Ca2+ sensors and play crucial roles in response to abiotic stress in plants. A salt-repressed PvCML9 from halophyte seashore paspalum (Paspalum vaginatum O. Swartz) was identified. PvCML9 was localized in the cytoplasm and nucleus and highly expressed in roots and stems. Overexpression of PvCML9 led to reduced salt tolerance in rice and seashore paspalum, whereas downregulating expression of PvCML9 showed increased salt tolerance in seashore paspalum as compared with the wild type (WT), indicating that PvCML9 regulated salt tolerance negatively. Na+ and K+ homeostasis was altered by PvCML9 expression. Lower level of Na+/K+ ratio in roots and shoots was maintained in PvCML9-RNAi lines compared with WT under salt stress, but higher level in overexpression lines. Moreover, higher levels of SOD and CAT activities and proline accumulation were observed in PvCML9-RNAi lines compared with WT under salt stress, but lower levels in overexpression lines, which altered ROS homeostasis. Based on the above data, mutation of its homolog gene OsCML9 in rice by CRISPR/Cas9 was performed. The mutant had enhanced salt tolerance without affecting rice growth and development, suggesting that OsCML9 gene is an ideal target gene to generate salt tolerant cultivars by genome editing in the future.

An upstream signaling gene calmodulin regulates the synthesis of insect wax via activating fatty acid biosynthesis pathway.

Insect wax accumulates on the surface of insect cuticle, which acts as an important protective barrier against rain, ultraviolet light radiation, pathogens, etc. The waxing behavior, wax composition and molecular mechanism underling wax biosynthesis are unclear in dustywings. Herein, the current study determined the vital developmental stage for waxing behavior in dustywings, examined the components of waxy secretions, and identified key regulatory genes for wax biosynthesis. The wax glands were mainly located on the thorax and abdomen of dustywing adults. The adults spread the waxy secretions over their entire body surface. The metabolomics analysis identified 32 lipids and lipid-like molecules, 15 organic acids and derivatives, 7 benzenoids, etc. as the main components of waxy secretions. The fatty acids represented the largest proportion of the category of lipid and lipid-like molecules. The conjoint analysis of metabolomics and transcriptomics identified two crucial genes fatty acyl-CoA reductase (CsFAR) and calmodulin (CsCaM) for wax biosynthesis. The down-regulation of these genes via nanocarrier-mediated RNA interference technology significantly reduced the amount of wax particles. Notably, the RNAi of CsCaM apparently suppressed the expression of most genes in fatty acid biosynthesis pathway, indicating the CsCaM might act as a main upstream regulator of fatty acid biosynthesis pathway.

MicroRNA1432 regulates rice drought stress tolerance by targeting the CALMODULIN-LIKE2 gene.

Due to climate change, drought has become a major threat to rice (Oryza sativa L.) growth and yield worldwide. Understanding the genetic basis of drought tolerance in rice is therefore of great importance. Here, we identified a microRNA, miR1432, which regulates rice drought tolerance by targeting the CALMODULIN-LIKE2 (OsCaML2) gene. Mutation of MIR1432 or suppression of miR1432 expression significantly impaired seed germination and seedling growth under drought-stress conditions. Molecular analysis demonstrated that miR1432 affected rice drought tolerance by directly targeting OsCaML2, which encodes an EF-hand chiral calcium-binding protein. Overexpression of a miR1432-resistant form of OsCaML2 (OEmCaML2) phenocopied the mir1432 mutant and miR1432 suppression plants. Furthermore, the suppression of miR1432 severely affected the expression of genes involved in responses to stimulation, metabolism and signal transduction, especially the mitogen-activated protein kinase (MAPK) pathway and hormone transduction pathway in rice under drought stress. Thus, our findings show that the miR1432-OsCaML2 module plays an important role in the regulation of rice drought tolerance, suggesting its potential utilization in developing molecular breeding strategies that improve crop drought tolerance.

CALMODULIN-LIKE16 and PIN-LIKES7a cooperatively regulate rice seedling primary root elongation under chilling.

Low-temperature sensitivity at the germination stage is a challenge for direct seeding of rice in Asian countries. How Ca2+ and auxin (IAA) signaling regulate primary root growth under chilling remains unexplored. Here, we showed that OsCML16 interacted specifically with OsPILS7a to improve primary root elongation of early rice seedlings under chilling. OsCML16, a subgroup 6c member of the OsCML family, interacted with multiple cytosolic loop regions of OsPILS7a in a Ca2+-dependent manner. OsPILS7a localized to the endoplasmic reticulum membranes and functioned as an auxin efflux carrier in a yeast growth assay. Transgenics showed that presence of OsCML16 enhanced primary root elongation under chilling, whereas the ospils7a knockout mutant lines showed the opposite phenotype. Moreover, under chilling conditions, OsCML16 and OsPILS7a-mediated Ca2+ and IAA signaling and regulated the transcription of IAA signaling-associated genes (OsIAA11, OsIAA23, and OsARF16) and cell division marker genes (OsRAN1, OsRAN2, and OsLTG1) in primary roots. These results show that OsCML16 and OsPILS7a cooperatively regulate primary root elongation of early rice seedlings under chilling. These findings enhance our understanding of the crosstalk between Ca2+ and IAA signaling and reveal insights into the mechanisms underlying cold-stress response during rice germination.

Redundant role of OsCNGC4 and OsCNGC5 encoding cyclic nucleotide-gated channels in rice pollen germination and tube growth.

In staple crops, such as rice (Oryza sativa L.), pollen plays a crucial role in seed production. However, the molecular mechanisms underlying rice pollen germination and tube growth remain underexplored. Notably, we recently uncovered the redundant expression and mutual interaction of two rice genes encoding cyclic nucleotide-gated channels (CNGCs), OsCNGC4 and OsCNGC5, in mature pollen. Building on these findings, the current study focused on clarifying the functional roles of these two genes in pollen germination and tube growth. To overcome functional redundancy, we produced gene-edited rice plants with mutations in both genes using the CRISPR-Cas9 system. The resulting homozygous OsCNGC4 and OsCNGC5 gene-edited mutants (oscngc4/5) exhibited significantly lower pollen germination rates than the wild type (WT), along with severely reduced fertility. Transcriptome analysis of the double oscngc4/5 mutant revealed downregulation of genes related to receptor kinases, transporters, and cell wall metabolism. To identify the direct regulators of OsCNGC4, which form a heterodimer with OsCNGC5, we screened a yeast two-hybrid library containing rice cDNAs from mature anthers. Subsequently, we identified two calmodulin isoforms (CaM1-1 and CaM1-2), NETWORKED 2 A (NET2A), and proline-rich extension-like receptor kinase 13 (PERK13) proteins as interactors of OsCNGC4, suggesting its roles in regulating Ca2+ channel activity and F-actin organization. Overall, our results suggest that OsCNGC4 and OsCNGC5 may play critical roles in pollen germination and elongation by regulating the Ca2+ gradient in growing pollen tubes.

Stripe rust effector Pst03724 modulates host immunity by inhibiting NAD kinase activation by a calmodulin.

Puccinia striiformis f. sp. tritici (Pst) secretes effector proteins that enter plant cells to manipulate host immune processes. In this report, we present an important Pst effector, Pst03724, whose mRNA expression level increases during Pst infection of wheat (Triticum aestivum). Silencing of Pst03724 reduced the growth and development of Pst. Pst03724 targeted the wheat calmodulin TaCaM3-2B, a positive regulator of wheat immunity. Subsequent investigations revealed that Pst03724 interferes with the TaCaM3-2B-NAD kinase (NADK) TaNADK2 association and thus inhibits the enzyme activity of TaNADK2 activated by TaCaM3-2B. Knocking down TaNADK2 expression by virus-mediated gene silencing significantly increased fungal growth and development, suggesting a decrease in resistance against Pst infection. In conclusion, our findings indicate that Pst effector Pst03724 inhibits the activity of NADK by interfering with the TaCaM3-2B-TaNADK2 association, thereby facilitating Pst infection.

Calmodulin-like protein CML15 interacts with PP2C46/65 to regulate papaya fruit ripening via integrating calcium, ABA and ethylene signals.

It is well known that calcium, ethylene and abscisic acid (ABA) can regulate fruit ripening, however, their interaction in the regulation of fruit ripening has not yet been fully clarified. The present study found that the expression of the papaya calcium sensor CpCML15 was strongly linked to fruit ripening. CpCML15 could bind Ca2+ and served as a true calcium sensor. CpCML15 interacted with CpPP2C46 and CpPP2C65, the candidate components of the ABA signalling pathways. CpPP2C46/65 expression was also related to fruit ripening and regulated by ethylene. CpCML15 was located in the nucleus and CpPP2C46/65 were located in both the nucleus and membrane. The interaction between CpCML15 and CpPP2C46/65 was calcium dependent and further repressed the activity of CpPP2C46/65 in vitro. The transient overexpression of CpCML15 and CpPP2C46/65 in papaya promoted fruit ripening and gene expression related to ripening. The reduced expression of CpCML15 and CpPP2C46/65 by virus-induced gene silencing delayed fruit colouring and softening and repressed the expression of genes related to ethylene signalling and softening. Moreover, ectopic overexpression of CpCML15 in tomato fruit also promoted fruit softening and ripening by increasing ethylene production and enhancing gene expression related to ripening. Additionally, CpPP2C46 interacted with CpABI5, and CpPP2C65 interacted with CpERF003-like, two transcriptional factors in ABA and ethylene signalling pathways that are closely related to fruit ripening. Taken together, our results showed that CpCML15 and CpPP2Cs positively regulated fruit ripening, and their interaction integrated the cross-talk of calcium, ABA and ethylene signals in fruit ripening through the CpCML15-CpPP2Cs-CpABI5/CpERF003-like pathway.

Molecular Analysis for Potential Hospital-Acquired Infection Caused by Aspergillus Tubingensis Through the Environment.

The identification of Aspergillus species has been performed mainly by morphological classification. In recent years, however, the revelation of the existence of cryptic species has required genetic analysis for accurate identification. The purpose of this study was to investigate five Aspergillus section Nigri strains isolated from a patient and the environment in a university hospital. Species identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry identified all five black Aspergillus strains as Aspergillus niger. However, calmodulin gene sequence analysis revealed that all five strains were cryptic species, four of which, including the clinical strain, were Aspergillus tubingensis. Hospital-acquired infection of the patient with the A. tubingensis strain introduced from the environment was suspected, but sequencing of six genes from four A. tubingensis strains revealed no environmental strain that completely matched the patient strain. The amount of in vitro biofilm formation of the four examples of the A. tubingensis strain was comparable to that of Aspergillus fumigatus. An extracellular matrix was observed by electron microscopy of the biofilm of the clinical strain. This study suggests that various types of biofilm-forming A. tubingensis exist in the hospital environment and that appropriate environmental management is required.