Calmodulin Research Articles

A new Paecilomyces from wooden utility poles in South Africa.

During a survey of fungi on electricity utility poles in South Africa, a diverse range of fungi were discovered. Paecilomyces was frequently isolated, with five species identified using ITS and β-tubulin (BenA) sequences. These were P. brunneolus, P. dactylethromorphus, P. lecythidis, P. paravariotii and a potential new species. The genomes of 30 of these strains were subsequently sequenced and used in a phylogenomic analysis with 45 previously published genomes of the genus. Phylogenetic analyses were also conducted using ITS, BenA, calmodulin (CaM), RNA polymerase II second largest subunit (RPB2), RNA polymerase II largest subunit (RPB1), the genes coding for the theta subunit of the TCP-1 chaperonin complex (Cct8), and for a putative ribosome biogenesis protein (Tsr1). Both phylogenomic and phylogenetic analyses supported the 15 Paecilomyces species currently accepted and confirmed the novelty of the new species, which we describe as P. lignorum. The latter is the sister species of P. brunneolus and belongs to a clade also containing P. variotii and P. paravariotii. Morphologically, the new species produces longer ellipsoidal conidia and grows more restricted on malt extract agar at 30 °C compared to its closest relatives. Citation: Visagie CM, Cruywagen EM, Duong TA (2024). A new Paecilomyces from wooden utility poles in South Africa. Fungal Systematics and Evolution 13: 163-181. doi: 10.3114/fuse.2024.13.10.

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.

Effects of β-asarone on spatial learning and memory impairment by exhaustive exercise-induced fatigue: Role of NR-CaMKII-ERK/CREB signal in hippocampus of rats

ObjectiveIt is to investigate the effects of β-asarone on learning and memory, hippocampal morphology, synaptophysin (SYP) and postsynaptic density 95(PSD95) protein expression, N-methyl-D-aspartic acid receptor 2B (NR2B)- Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) - Extracellular signal-regulated kinase (ERK) / Cyclic-AMP response element binding protein (CREB) signal in hippocampus of rats with exhaustive exercise-induced fatigue. MethodsFifty Sprague-Dawley male rats were randomly divided into five groups: normal group, exercise group, exercise and β-asarone (2.5, 10, 40 mg/kg)–treated groups. The learning and memory in rats were tested by Morris water maze experiment. We measured the hippocampal morphology by Nissl staining. The levels of SYP, PSD95, NR2B, CaMKII, ERK1/2, CREB, p-NR2B, p-CaMKII, p-ERK1/2 and p-CREB expression were measured by western blot analysis. ResultsThe results demonstrated that β-asarone (10, 40 mg/kg) treatment significantly decreased the latency to find the platform, increased the time spent in the target quadrant and the number of crossing the platform of rats with exhaustive exercise-induced fatigue. β-asarone (10, 40 mg/kg) treatment increased the cell density in the hippocampus CA1 region, significantly up-regulated NR2B-CaMKII-ERK/CREB signal and improved the protein expression levels of SYP and PSD95 in hippocampus of rats with exhaustive exercise-induced fatigue. ConclusionsIt suggests that β-asarone could improve learning and memory of rats with exhaustive exercise-induced fatigue. The mechanism might be related to β-asarone protecting the morphology of hippocampus, increasing the protein expression levels of SYP and PSD95 and up-regulating NR2B-CaMKII-ERK/CREB signal in hippocampus of rats with exhaustive exercise-induced fatigue.

Characterization of Colletotrichum fructicola causing anthracnose on Schima superba in China

AbstractLeaves of Schima superba with typical anthracnose symptoms were collected in July 2023 in Zhejiang Province, China. The fungi isolates were conducted from the margins of the lesions, and three isolates CA13, CA18 and CA27, were selected for identification and pathogenicity test. Based on morphological characteristics and phylogenetic analyses of the partial regions of internal transcribed spacer (ITS), actin (ACT), calmodulin (CAL), beta‐tubulin (TUB2), and glyceraldehyde‐3‐phosphate‐dehydrogenase sequences (GAPDH), the fungi were identified as Colletotrichum fructicola. Inoculation of the fungi on healthy S. superba leaves showed symptoms similar to those observed in the field, and the fungi were re‐isolated from the lesions. To the best of our knowledge, this represents the first report of C. fructicola causing anthracnose on S. superba worldwide.

Calmodulin‐Based Dynamic Protein Hydrogels with Three Distinct Mechanical Stiffness

AbstractStimuli‐responsive hydrogels that leverage protein conformational changes are of significant interest in the design of dynamic materials applicable in a myriad of fields, such as drug delivery, actuators, biosensors, and microfluidics. The small calcium binding protein calmodulin (CaM), which undergoes three‐stage conformational changes upon successive binding with Ca2+ and specific ligands, offers a mechanism to create dynamic hydrogels with three distinct physical states. In this work, a CaM‐based recombinant protein hydrogel is engineered using [Ru(bpy)3]2+‐mediated photo‐crosslinking. This hydrogel displays the ability to reversibly increase its Young's modulus by 1.5‐fold and ∼7‐fold, respectively, upon binding with Ca2+ and subsequent interaction with trifluoperazine. The magnitude of stiffness changes is tunable by adjusting the length proportion of dynamic and static domains and modifying protein content. This tunable and reversible control over hydrogel mechanics is further utilized to engineer shape‐morphing materials, highlighting the versatile potential of this CaM‐based protein hydrogel for diverse applications.

Salt gradient enhanced sensitivity in nanopores for intracellular calcium ion detection

Intracellular calcium ion detection is of great significance for understanding the cell metabolism and signaling pathways. Most of the current ionic sensors either face the size issue or sensitivity limit for the intracellular solution with high background ion concentrations. In this paper, we proposed a calmodulin (CaM) functionalized nanopore for sensitive and selective Ca2+ detection inside living cells. A salt gradient was created when the nanopore sensor filled with a low concentration electrolyte was in contact with a high background concentration solution, which enhanced the surface charge-based detection sensitivity. The nanopore sensor showed a 10 × sensitivity enhancement by application of a 100-fold salt gradient, and a detection limit of sub nM. The sensor had a wide detection range from 1 nM to 1 mM, and allowed for quick calcium ion quantification in a few seconds. The sensor was demonstrated for intracellular Ca2+ detection in A549 cells in response to ionomycin.

Mapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry.

Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain-domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., Biochemistry, 2023, 62, 2232-2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodimeric nNOS oxygenase/FMN (oxyFMN) construct, an established model of the NOS output state. We employed parallel reaction monitoring (PRM) based quantitative XL MS (qXL MS) to assess the CaM-induced changes in interdomain dynamics and interactions. Intersubunit cross-links were identified by mapping the cross-links onto top AlphaFold structural models, which was complemented by comparing their relative abundances in the cross-linked dimeric and monomeric bands. Furthermore, contrasting the CaM-free and CaM-bound nNOS samples shows that CaM enables the formation of the intersubunit FMN-heme docking complex and that CaM binding induces extensive, allosteric conformational changes across the NOS regions. Moreover, the observed cross-links sites specifically respond to changes in ionic strength. This indicates that interdomain salt bridges are responsible for stabilizing and orienting the output state for efficient FMN-heme IET. Taken together, our targeted qXL MS results have revealed that CaM and ionic strength modulate specific dynamic changes in the CaM/FMN/heme complexes, particularly in the context of intersubunit interdomain FMN-heme interactions.

Neurogranin modulates the rate of association between calmodulin and target peptides

The best-known mode of action of calmodulin (CaM) is binding of Ca2+ to its N- and C-domains, followed by binding to target proteins. An underappreciated facet of this process is that CaM is typically bound to proteins at basal levels of free Ca2+, including the small, intrinsically disordered, neuronal IQ-motif proteins called PEP-19 and neurogranin (Ng). PEP-19 and Ng would not be effective competitive inhibitors of high-affinity Ca2+-dependent CaM targets at equilibrium because they bind to CaM with relatively low affinity, but they could influence the time course of CaM signaling by affecting the rate of association of CaM with high-affinity Ca2+-dependent targets. This mode of regulation may be domain specific because PEP-19 binds to the C-domain of CaM, whereas Ng binds to both N- and C-domains. In this report, we used a model CaM binding peptide (CKIIp) to characterize the preferred pathway of complex formation with Ca2+-CaM at low levels of free Ca2+ (0.25–1.5 μM), and how PEP-19 and Ng affect this process. We show that the dominant encounter complex involves association of CKIIp with the N-domain of CaM, even though the C-domain has a greater affinity for Ca2+. We also show that Ng greatly decreases the rate of association of Ca2+-CaM with CKIIp due to the relatively slow dissociation of Ng from CaM, and to interactions between the Gly-rich C-terminal region of Ng with the N-domain of CaM, which inhibits formation of the preferred encounter complex with CKIIp. These results provide the general mechanistic paradigms that binding CaM to targets can be driven by its N-domain, and that low-affinity regulators of CaM signaling have the potential to influence the rate of activation of high-affinity CaM targets and potentially affect the distribution of limited CaM among multiple targets during Ca2+ oscillations.

Zinc Oxide:Gold Nanoparticles (ZnO:Au NPs) Exhibited Antifungal Efficacy Against Aspergillus niger and Aspergillus candidus

Fungal pathogens are a major health issue that threatens the era of antifungal drugs commonly used in the treatment of infections. An effective approach of biosynthetic nanoparticles can be used as antifungal agents owing to their intrinsic features such as their simplicity, non-toxic, and physicochemical properties. Therefore, this study was aimed to molecularly ascertain Aspergillus species known to cause aspergillosis and investigate the potency of zinc oxide:gold nanoparticles (ZnO:Au NPs) against the fungal pathogens. Two Aspergillus strains retrieved with potato dextrose agar (PDA) culture media from commercial food products in South Africa were molecularly identified using calmodulin (CaM) gene region. DNA sequence phylogeny of the gene showed that the strains were A. niger and A. candidus. ZnO:Au (1%) NPs were synthesised and characterised using X-ray diffraction (XRD), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS). Two distinct peak plasmon bands for ZnO and ZnO:Au NPs were 390 nm and 565 nm, respectively. FE-SEM images demonstrated the presence of Au on the surface of ZnO nanoparticles in the ZnO:Au nanocomposites. The ZnO:Au NPs antifungal activity of 10 µg/mL and 50 µg/mL concentrations were evaluated against the two Aspergillus spp. ZnO:Au NPs at 50 µg/mL exhibited a maximum antifungal activity against A. candidus and A. niger, with zones of inhibition (ZoI) of 31.2 ± 0.15 mm and 25.0 ± 0.06, respectively. When the ZoI was observed by SEM, major morphological damages on the conidia were observed for both strains, indicating that the antifungal activity may have been enhanced by the ZnO:Au NPs. Therefore, due to these outstanding properties, ZnO:Au NPs can be utilised as potential antifungal agents to inhibit the proliferation of fungal pathogens.

Occurrence of Cassava (Manihot esculenta) Leaf Spot Disease Caused by Diaporthe ueckeri in China.

Cassava (Manihot esculenta) is a perennial crop of the family Euphorbiaceae, widely cultivated due to its phytopharmacological and economic values in China. In November 2022, a leaf spot disease on cassava was observed in Zhanjiang, Guangdong, China (21.17° N, 110.18° E), with 100% disease incidence. About 80 % of leaves were covered with spots on the infected plants. Typical symptoms initially appeared as irregular water-soaked lesions that became brown and whitish with the progress of the disease, lesions gradually expanded and coalesced, causing leaf withering, drying and final fall. Tissues (4 to 5 mm) were excised from the margin of lesions, sterilized in 3% H2O2 solution for 3 min, rinsed three times with sterile water, placed on potato dextrose agar (PDA) medium (containing 50mg/L penicillin), and incubated at 25-28 °C. Ten single hypha isolates with similar morphology were obtained and further purified as single conidium subcultures. The colony was grey whitish with sparse aerial mycelium and colony diameter reached 70.4 mm after four days incubation at 25-28℃ in the dark. Black pycnidia occurring as clusters were spherical or irregular, erumpent at maturity, often with a creamy whitish, conidial cirrus extruding from ostiole after 30-days incubation. Conidiophores were hyaline, smooth, unbranched. Alpha conidia were bi-guttulate, hyaline, ellipsoidal, aseptate, with dimensions of 5.1~7.5×1.9~3.4µm (mean 6.2×2.8 µm, n>50). Beta conidia were abundant, filiform, hyaline, smooth, curved in a hooked shape, with a truncate base and dimensions of 18.5-26.4 × 0.6-1.2μm (mean 23.4 × 1.0 μm, n= 40) . Gamma conidia were not observed. The morphological characteristics were similar to those of Diaporthe ueckeri (Udayanga et al. 2015). The internal transcribed spacer (ITS) region, large subunit (LSU) rRNA sequence, actin (ACT), calmodulin (CAL), histone H3 (HIS), translation elongation factor 1-alpha (TEF1-α), and β-tubulin (TUB) genes of a representative isolate CCAS-MS-6 (ACCC 35497) were amplified and sequenced using primer pairs: ITS5/ITS4, LR0R/LR5, ACT-512F/ACT-783R, CAL228F/CAL737R, CYLH3F/ H3-1b, EF1-728F/ EF1-986R and Bt2a/Bt2b (Gao et al 2017；Udayanga et al 2014). All sequences were deposited in GenBank (OR361671, OR361672, and OR365605-9). BLAST search showed high similarities with sequences of Diaporthe ueckeri (Tab 1). Maximum likelihood analyses of the concatenated data of CAL, HIS, ITS, TEF and TUB using Mega 11 placed CCAS-MS-6 in the D. ueckeri clade. Thus, the fungus was identified as D. ueckeri. Three one-year old healthy plants were used for pathogenicity tests in pots. Two 15-day old leaves of each plant were cleaned with 75% alcohol, three sites on each leaf were wounded, and sites on one of the leaf were covered with fungal plugs from 15-day-old cultures on PDA, and sites on the other leaf with PDA plugs as a control. All plants were kept at ambient temperature (about 28℃) and covered with plastic bags containing sterile wet cotton to maintain the humidity. Seven days after inoculation, all inoculated sites showed symptoms of necrosis, while control sites showed no symptoms. The same fungus identified on the basis of morphological and molecular criteria was reisolated from symptomatic inoculated leaves. In China, D. ueckeri had been reported to cause diseases on Eucalyptus citriodora, Camellia sinensis, and Michelia shiluensis (Gao et al 2016; Liao et al 2023; Yi et al 2018), this is the first report on M. esculenta. The definition of the disease etiology is a prerequisite to develop effective management strategies.

Heat stress-induced NO enhanced perylenequinone biosynthesis of Shiraia sp. via calcium signaling pathway

Perylenequinones (PQs) are natural photosensitizing compounds used as photodynamic therapy, and heat stress (HS) is the main limiting factor of mycelial growth and secondary metabolism of fungi. This study aimed to unravel the impact of HS-induced Ca2+ and the calcium signaling pathway on PQ biosynthesis of Shiraia sp. Slf14(w). Meanwhile, the intricate interplay between HS-induced NO and Ca2+ and the calcium signaling pathway was investigated. The outcomes disclosed that Ca2+ and the calcium signaling pathway activated by HS could effectively enhance the production of PQs in Shiraia sp. Slf14(w). Further investigations elucidated the specific mechanism through which NO signaling molecules induced by HS act upon the Ca2+/CaM (calmodulin) signaling pathway, thus propelling PQ biosynthesis in Shiraia sp. Slf14(w). This was substantiated by decoding the downstream positioning of the CaM/CaN (calcineurin) pathway in relation to NO through comprehensive analyses encompassing transcript levels, enzyme assays, and the introduction of chemical agents. Concurrently, the engagement of Ca2+ and the calcium signaling pathway in heat shock signaling was also evidenced. The implications of our study underscore the pivotal role of HS-induced Ca2+ and the calcium signaling pathway, which not only participate in heat shock signal transduction but also play an instrumental role in promoting PQ biosynthesis. Consequently, our study not only enriches our comprehension of the mechanisms driving HS signaling transduction in fungi but also offers novel insights into the PQ synthesis paradigm within Shiraia sp. Slf14(w).Key points• The calcium signaling pathway was proposed to participate in PQ biosynthesis under HS.• HS-induced NO was revealed to act upon the calcium signaling pathway for the first time.

Phytosanitary problems in elephant garlic (Allium ampeloprasum var. holmense) in the “Val di Chiana” area (Central Italy), and evaluation of potential control strategies

Allium ampeloprasum var. holmense (elephant garlic) is traditionally cultivated in “Val di Chiana”, an area between Umbria and Tuscany regions of Central Italy, under the name “Aglione della Valdichiana”. This product has recently increased in importance, becoming a key economic resource for local farmers. In 2019, phytosanitary problems of elephant garlic cloves ready for transplanting emerged in this cultivation area. Symptom/sign observations and fungal isolations were performed for cloves divided into four components (tunic, basal plate, reserve tissue and shoot) from six farms in the “Val di Chiana” area. Isolates obtained were identified, using partial β-tubulin (BenA) and calmodulin (CaM) or translation elongation factor 1α (tef1α) genes sequences, as belonging to Penicillium [P. allii (95%), P. citrinum (4%), P. brevicompactum (1%)] or Fusarium [F. oxysporum (81%), F. proliferatum (19%)]. Fusarium spp. were mainly associated with clove tunics and basal plates, while Penicillium spp. with basal plates, reserve tissues and shoots. Fungi often also developed from asymptomatic components, but a correlation was found between isolated pathogens and disease symptoms. Pathogenicity and virulence towards elephant garlic cloves were verified for a representative isolate of each identified species, and Penicillium allii was the most virulent. Strategies to control Fusarium and Penicillium spp. on cloves were assessed, including chemicals, a biocontrol agent, surface sterilization and heat treatment. Among these, treatments with Patriot Gold® (active ingredient [a.i.] Trichoderma asperellum TV1, approved in organic farming on crops similar to elephant garlic), or Signum® (a.i. boscalid + pyraclostrobin, approved for Integrated Pest Management systems on crops similar to elephant garlic), were effective in simultaneous reduction of Penicillium spp. and Fusarium spp. Transplanting of asymptomatic cloves combined with the use of the above treatments showed promising effects for pathogens control, and to assist elephant garlic crop establishment.

Development of a TaqMan Real-Time PCR for Early and Accurate Detection of Anthracnose Pathogen Colletotrichum siamense in Pachira glabra.

Anthracnose, caused by Colletotrichum siamense, is a destructive disease of Pachira glabra in southern China. Early and proper monitoring and quantification of C. siamense is of importance for disease control. A calmodulin (CAL) gene-based TaqMan real-time PCR assay was developed for efficient detection and quantification of C. siamense, which reliably detected as low as 5 pg of genomic DNA and 12.8 fg (5800 copies) of target DNA. This method could specifically recognize all tested C. siamense isolates, while no amplification was observed in other closely related Colletotrichum species. The assay could still detect C. siamense in plant mixes, of which only 0.01% of the tissue was infected. A dynamic change in the amount of C. siamense population was observed during infection, suggesting that this real-time PCR assay can be used to monitor the fungal growth progression in the whole disease process. Moreover, the method enabled the detection of C. siamense in naturally infected and symptomless leaves of P. glabra trees in fields. Taken together, this specific TaqMan real-time PCR provides a rapid and accurate method for detection and quantification of C. siamense colonization in P. glabra, and will be useful for prediction of the disease to reduce the epidemic risk.

Fungal alkaloid malbrancheamide reorients the lipid binding domain of GRK5

G protein-coupled receptors (GPCRs) are the largest group of receptors involved in various types of signaling. GPCR signaling is regulated via receptor phosphorylation by G protein-coupled receptor kinases 5 (GRK5). Calmodulin (CaM), a universal Ca2+ sensor, inhibits receptor phosphorylation by binding to GRK5. However, the inhibitor malbrancheamide (MBC), which binds at CaM C-lobe, allows for receptor phosphorylation. To understand the phosphorylation mechanism by GRK5, we carried out a MD simulation of the CaM/GRK5 complex in the presence and absence of the MBC inhibitor. The lipid binding domain (LBD) of GRK5 adopted different positions in the presence and absence of inhibitor. Furthermore, the inhibitor MBC restricted the movement of the N-lobe tether (NLT) loop, probably blocking the autophosphorylation of GRK5. Communicated by Ramaswamy H. Sarma

Investigating the Impact of Electrostatic Interactions on Calmodulin Binding and Ca2+-Dependent Activation of the Calcium-Gated Potassium SK4 Channel.

Ca2+ binding to the ubiquitous Ca2+ sensing protein calmodulin (CaM) activates the intermediate conductance Ca2+-activated SK4 channel. Potential hydrophilic pockets for CaM binding have been identified at the intracellular HA and HB helices in the C-terminal of SK4 from the three published cryo-EM structures of SK4. Single charge reversal substitutions at either site, significantly weakened the pull-down of SK4 by CaM wild-type (CaM), and decreased the TRAM-34 sensitive outward K+ current densities in native HEK293T cells when compared with SK4 WT measured under the same conditions. Only the doubly substituted SK4 R352D/R355D (HB helix) obliterated the CaM-mediated pull-down and thwarted outward K+ currents. However, overexpression of CaM E84K/E87K, which had been predicted to face the arginine doublet, restored the CaM-mediated pull-down of SK4 R352D/R355D and normalized its whole-cell current density. Virtual analysis of the putative salt bridges supports a unique role for the positively charged arginine doublet at the HB helix into anchoring the interaction with the negatively charged CaM glutamate 84 and 87 CaM. Our findings underscore the unique contribution of electrostatic interactions in carrying CaM binding onto SK4 and support the role of the C-terminal HB helix to the Ca2+-dependent gating process.

Disruption of Ca2+/calmodulin:KSR1 interaction lowers ERK activation.

KSR1, a key scaffold protein for the MAPK pathway, facilitates ERK activation upon growth factor stimulation. We recently demonstrated that KSR1 binds the Ca2+-binding protein calmodulin (CaM), thereby providing an intersection between KSR1-mediated and Ca2+ signaling. In this study, we set out to generate a KSR1 point mutant with reduced Ca2+/CaM binding in order to unravel the functional implications of their interaction. To do so, we solved the structural determinants of complex formation. Using purified fragments of KSR1, we showed that Ca2+/CaM binds to the CA3 domain of KSR1. We then used in silico molecular modeling to predict contact residues for binding. This approach identified two possible modes of interaction: (1) binding of extended Ca2+/CaM to a globular conformation of KSR1-CA3 via electrostatic interactions or (2) binding of collapsed Ca2+/CaM to α-helical KSR1-CA3 via hydrophobic interactions. Experimentally, site-directed mutagenesis of the predicted contact residues for the two binding models favored that where collapsed Ca2+/CaM binds to the α-helical conformation of KSR1-CA3. Importantly, replacing KSR1-Phe355 with Asp reduces Ca2+/CaM binding by 76%. The KSR1-F355D mutation also significantly impairs the ability of EGF to activate ERK, which reveals that Ca2+/CaM binding promotes KSR1-mediated MAPK signaling. This work, by uncovering structural insight into the binding of KSR1 to Ca2+/CaM, identifies a KSR1 single-point mutant as a bioreagent to selectively study the crosstalk between Ca2+ and KSR1-mediated signaling.

First report of leaf blight caused by Stemphylium lycopersici on Salvia splendens in China.

Salvia splendens is a popular ornamental plant in China with extensive potentials, including value in traditional Chinese medicine and in environmental restoration function (Li et al. 2008). In September 2019, leaf blight disease was observed on road side plants of S. splendens in Bayi park, Nanchang city, Jiangxi province, China. The typical symptoms appeared as irregular necrotic spots or leaf blight, accompanied by extensive scorch necrosis or ultimately defoliation. Small segments cut from diseased leaves were surface sterilized in a 2% sodium hypochlorite solution for 2 min and rinsed three times with sterile distilled water. Then, the samples were placed on potato dextrose agar (PDA) plates incubated at 25°C in darkness. Pure cultures were obtained by the hyphal tip method. Morphologically, all 11 colonies were identical to each other on PDA. Two strains, YZU 191468 and YZU 191481, were selected for further study and deposited in the Fungal Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China. The 7-day-old colonies were circular, 53 to 56 mm in diameter, and consisted of white mycelium with a buff margin, and were cinnamon colored in the center of the reverse side. To examine conidial morphology, the mycelium was transferred onto potato carrot agar (PCA) and incubated at 23°C with a period of 8 h light/16 h dark for 7 days. Conidia were normally solitary or two in a chain, ellipsoid or long ellipsoid, beakless, 10 to 23×30 to 60 µm in size (n=50). Based on morphology, the isolates were consistent with Stemphylium lycopersici (Yamamoto 1960). To confirm the identification, genomic DNA was extracted from both isolates and used to amplify the internal transcribed spacer rDNA region (ITS), glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and calmodulin (CAL) genes with primer pairs ITS5/ITS4, gpd1/gpd2, and CALDF1/CALDR2, respectively (Woudenberg et al. 2017). Sequences were deposited in GenBank with accession numbers OP564983 and OP564984 (ITS), OP892529 and OP892530 (GAPDH), OP584970 and OP584971 (CAL). A neighbor-joining tree was constructed with Mega 7.0 based on the combined dataset with 1,000 bootstrap replicates. The resulting phylogenetic tree showed that the strains from S. splendens clustered with S. lycopersici (CBS 122639 and CBS 124980) supported with 100% bootstrap values. The molecular analyses confirmed that the species causing leaf blight symptoms was S. lycopersici. To test pathogenicity, healthy leaves of S. splendens were surface sterilized and inoculated by mycelium blocks (6 mm in diameter) and spore suspension (1×106 spore/mL) of representative strains YZU 191468 and YZU 191481, respectively. Controls were inoculated with blocks of PDA and sterile water. Each strain was inoculated on three leaves of a plant. One clean plant was used as control. The test was replicated three times. After inoculation, the plants were covered with plastic bags and incubated in a greenhouse (25℃, 80 % relative humidity, 8 h light/16 h dark). After 5 days, the inoculated leaves exhibited dark brown spots with white mycelium, followed by withering of necrotic tissues. There were no symptoms observed on the controls. The fungal isolates inoculated leaves had the same morphological characteristics as the strains used for inoculation. S. lycopersici has been found on eggplant and Zinnia elegans in China (He et al. 2019; Yang et al. 2017). To the best of our knowledge, this is the first report of S. lycopersici causing leaf blight on S. splendens in China. This finding offers a new reference for the management and control of S. splendens leaf diseases in China.

Probing calmodulin-NO synthase interactions via site-specific infrared spectroscopy: an introductory investigation.

Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functionalconformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM-NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and abi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS). Site-specific, selective labeling with p-cyano-L-phenylalanine (CNF) by amber suppression of CaM bound to the iNOS has been accomplished by protein coexpression due to the instability ofrecombinant iNOS proteinalone. We introduced CNF at residue 108, which is at the putative CaM-heme (NOS) docking interface. CNF was also introduced at residue 29, which is distant from the docking interface. FT IR data show that the 108 site is sensitive to CaM-NOS complex formation, while insensitivity to its association with the iNOS protein or peptide was observed for the 29 site. Moreover, narrowing of the IR bands at residue 108 suggests the C≡N probe experiences a more limited distribution of environments, indicating side chain restriction apparent for the complex with iNOS. This initial work sets the stage for residue-specific characterizations of structural dynamics of the docked statesof NOS proteins.

Fusarium pernambucanum Causing Peduncle Rot on Grape in Guangxi, China.

Grape (Vitis vinifera) is regarded as one of the most economically important fruit crops throughout the world and is widely cultivated in China. In March 2023, peduncle rot symptoms were observed on ripe grape (cv. Kyoho) of Guangxi Academy of Agricultural Sciences in Nanning, Guangxi Province (20°54' to 26°24' N, 104°26' to 112°04' E), causing severe shrink of infected grapes with incidence of 20 to 40%. Initially, a small portion of the grape peduncles turned from light brown to darker brown and gradually spread to the entire bunch of grape peduncles. Then the peduncles gradually dried up and became dark brown, resulting in necrosis and shedding of the fruits. Symptomatic peduncles were cut into small pieces (approximately 3 mm long), surface disinfected with 75% alcohol for 10 s, 1% NaOCl for 2 min followed by three washes in sterile distilled water and separately transferred to potato dextrose agar (PDA) plates. Forty-six Fusarium isolates were obtained from the seventy-five tissue pieces after 5 days of incubation at 25°C on PDA (average isolation frequency 61%). Based on the morphology, a representative isolate of Fusarium was selected for each of the three samples. Single spore isolates (PT1-1, PT3-1, and PT3-2) were selected for further study. The colonies produced abundant whitish to yellowish aerial mycelium on PDA after 7 days incubation at 25°C in the dark. Macroconidia cultured on carnation leaf agar (CLA) for 7 days were falcate, multiseptate, with a curved apical cell and foot-shaped basal cell, mostly 1-3-septate, measuring 21.3 ± 0.5 μm × 4.8 ± 0.1 μm, 24.3 ± 0.5 μm × 4.5 ± 0.1 μm, 21.9 ± 0.3 μm × 4.5 ± 0.1 μm (n=90) for PT1-1, PT3-1, and PT3-2, respectively. Microconidia were hyaline, fusoid or ovoid, 0- or 1-septate, measuring 10.5 ± 0.2 μm × 3.5 ± 0.1 μm, 10.4 ± 0.3 μm × 3.6 ± 0.1 μm, 10.0 ± 0.2 μm × 3.6 ± 0.1 μm (n=90) for PT1-1, PT3-1, and PT3-2, respectively. The internal transcribed spacer (ITS), translation elongation factor (TEF1), calmodulin (CAM) and partial RNA polymerase second largest subunit (RPB2) were amplified using primers ITS4/ITS1, EF1/EF2, CL1/CL2A, and 5F2/7cR, respectively (White et al. 1990; O'Donnell et al. 1998, 2000; Reeb et al. 2004; Liu et al. 1999). Sequences from the three isolates were deposited in GenBank (ITS: OR511756-OR511758; TEF1: OR535156-OR535158; CAM: OR535153-OR535155; RPB2: OR535159-OR535161). A maximum likelihood (ML) phylogenetic tree was constructed with RAxML version 8.2.10 based on the concatenated sequences (ITS, CAM, TEF1, RPB2). According to morphology and phylogenetic analysis, the isolates were identified as Fusarium pernambucanum (Santos et al. 2019). A pathogenicity test was performed with three isolates on twenty-four asymptomatic strings of grapes in a field in Nanning, Guangxi Province, China. On twelve strings of ripe grapes (cv. Kyoho), 3-mm-long wounds were made on the peduncle of each string of grapes with a sterile needle, followed by inoculation with conidial suspension (106spores/ml in 0.1% sterile Tween 20) by misting with an atomizer until runoff (Lorenz et al. 1995). Three isolates were separately inoculated onto three strings of grapes. Controls were inoculated with water containing 0.1% sterile Tween 20 under the same conditions. The same inoculation was applied to twelve strings of non-wound grapes. Plants were covered with polythene bags to maintain high humidity for 7 days. Typical dark brown lesions were observed on all inoculated peduncles with conidia. After 14 days, the necrotic lesions spread to the entire peduncles causing them to shrivel and die. No symptoms were observed on controls. Koch's postulates were completed by reisolating the fungus from the inoculated tissues. The results of morphological identification and multigene sequence analysis obtained by reisolation were consistent with original isolates. To our knowledge, this is the first report of F. pernambucanum causing peduncle rot on grape in China. These results will provide valuable information for prevention and management of peduncle rot on grape.

First Report of Shoot Blight of Grapevine Caused by Sclerotinia sclerotiorum in Illes Balears, Mallorca, Spain.

In 2021, grapevines (Vitis vinifera L.) cv. Callet growing in a commercial vineyard located at Pollença (northeast of the island of Majorca, Spain) showed severe symptoms of shoot blight during spring and early summer, with an incidence of 70%. Symptoms consisted of elongated cankered-like lesions, surrounded by water-soaked darker tissues, that developed at the base or around the middle nodes of the shoot. For fungal isolation, shoot samples with lesions were collected, surface disinfected with 2% NaCl for 90s, rinsed twice with deionized water and placed in Petri plates containing potato dextrose agar (PDA). The plates were incubated at 25°C under 12 h light-darkness for 6 days. Isolations consistently yielded on kind of fungal colonies that produced white mycelium and black spherical to elongated sclerotia (2 to 10 mm in diameter). Morphological characterization was consistent with the description of Sclerotinia sclerotiorum (Lib.) de Bary (Bolton et al. 2006). Three isolates (UIB 118-1, UIB 118-26, and UIB 129-41) were preserved and deposited in the Culture Collection of Microbiology-Faculty of Sciences, University of Balearic Islands, Spain. Genomic DNA was extracted from isolates UIB 118-26 and UIB 129-41 using the EZNA Miniprep Kit (Omega Bio-Tek, Norcross, GA). The internal transcribed spacer (ITS) region of ribosomal DNA, β-tubulin (BTUB) and calmodulin (CAL) gene regions were amplified using ITS1F-ITS4 (Gardes and Bruns, 1996; White et al. 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995) and CAL228F/CAL737R (Carbone and Kohn 1999) primer sets, respectively. Amplicons were sequenced and deposited in GenBank with accession numbers MZ604647 and MZ604648 for ITS, OK634402 and OK634403 for BTUB and OK634404 and OK634405 for CAL. BLASTn search showed that isolates were >99 % (ITS, BTUB and CAL) identical to S. sclerotiorum GenBank accession no. KF859933, CP017815 and KF871381, respectively. Pathogenicity tests were conducted using eight one-year old grapevines cv. Cabernet Sauvignon. Old and new green shoots were inoculated by inserting a 6-mm plug of mycelium taken from actively growing cultures on PDA into cuts made at the base and at the distal part of each shoot with a sterile scalpel with a total of eight inoculation points per plant. Inoculated wounds were sealed with Parafilm tape to avoid rapid dehydration. Inoculated plants and an equal number of wounded but non-inoculated plants (negative controls) were maintained at 25 ± 1°C for 48 h in plastic containers to ensure a high relative humidity (>90%). After 5 days, the infection girdled and rotted the green new shoots, whereas the older partially lignified shoots developed a localized long brown lesion that reached 16 cm in length. Due to the rotting of the basal part of the petiole, leaves turned gray, wilted, and died, easily detaching from the stem. In advanced stages of the disease, 7 days after infection, branches died and fell with the leaves remained attached (Fig 1 A, B). Reisolations from diseased shoots were successfully performed on PDA to fulfill Koch's postulates. S. slerotiorum was previously reported on grapevine causing shoot blight in Chile (Latorre and Guerrero, 2001), Korea (Jong-Han et al. 2009), California-USA (Boland and Hall, 1994) and Australia (Hall et al. 2002). AlsoS. sclerotiorum was reported among the endophytic mycobiota associated with Vitis vinifera in the Iberian Peninsula (Gonzalez and Tello, 2011) but not as a pathogen causing visible symptoms on that crop. So, this is the first report of the occurrence of S. slerotiorum as a pathogen of grapevines in Spain causing symptoms of canker and shoot blight. This finding highlights a potential risk of this fungal disease for the wine industry in the Mediterranean region and specially for Spain, the country with the largest acreage devoted to grapevines. Although chemical and biological are suitable control strategies, disease management is difficult as sclerotia of Sclerotinia can remain in the soil for up to eight years (Adams and Ayears, 1979), and preventive surveys are greatly recommended as an important epidemiological tool to monitor the epidemiology of disease and identify potential outbreaks of this new pathogen on grapevine in Spain.