The P1 protein of wheat yellow mosaic virus exerts RNA silencing suppression activity to facilitate virus infection in wheat plants.

Wheat yellow mosaic virus (WYMV) causes severe wheat viral disease in Asia. However, the viral suppressor of RNA silencing (VSR) encoded by WYMV has not been identified. Here, the P1 protein encoded by WYMV RNA2 was shown to suppress RNA silencing in Nicotiana benthamiana. Mutagenesis assays revealed that the alanine substitution mutant G175A of P1 abolished VSR activity and mutant Y10A VSR activity remained only in younger leaves. P1, but not G175A, interacted with gene silencing-related protein, N. benthamiana calmodulin-like protein (NbCaM), and calmodulin-binding transcription activator 3 (NbCAMTA3), and Y10A interacted with NbCAMTA3 only. Competitive Bimolecular fluorescence complementation and co-immunoprecipitation assays showed that the ability of P1 disturbing the interaction between NbCaM and NbCAMTA3 was stronger than Y10A, Y10A was stronger than G175A. In vitro transcript inoculation of infectious WYMV clones further demonstrated that VSR-defective mutants G175A and Y10A reduced WYMV infection in wheat (Triticum aestivum L.), G175A had a more significant effect on virus accumulation in upper leaves of wheat than Y10A. Moreover, RNA silencing, temperature, and autophagy have significant effects on the accumulation of P1 in N. benthamiana. Taken together, WYMV P1 acts as VSR by interfering with calmodulin-associated antiviral RNAi defense to facilitate virus infection in wheat, which has provided clear insights into the function of P1 in the process of WYMV 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.

Oomycete effector AVRblb2 targets cyclic nucleotide-gated channels through calcium sensors to suppress pattern-triggered immunity.

Transient and rapid increase in cytosolic Ca2+ plays a crucial role in plant-pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Cyclic nucleotide-gated channels (CNGCs) have been implicated in mediating this Ca2+ influx; however, their regulatory mechanisms remain poorly understood. Here, we have found that AVRblb2 requires the calmodulin (CaM) and calmodulin-like (CML) proteins as co-factors to interact with the NbCNGCs, resulting in the formation of AVRblb2-CaM/CML-NbCNGCs complex. Furthermore, CaM and CML are dissociated from NbCNGC18 during PTI response to increase Ca2+ influx; however, Avrblb2 inhibits calcium channel activation by disrupting the release of CaM and CML from NbCNGC18. Following recognition of PAMP, NbCNGC18 forms active heteromeric channels with other NbCNGCs, which may give selectivity of CNGC complex against diverse signals for fine-tuning of cytosolic Ca2+ level to mediate appropriate responses. Silencing of multiple NbCNGCs compromised the function of AVRblb2 on the pathogenicity of Phytophthora infestans, confirming that AVRblb2 contributes to pathogen virulence by targeting CNGCs. Our findings provide new insights into the regulation of CNGCs in PTI and the role of pathogen effectors in manipulating host cell physiology to promote infection.

The nematode effector calreticulin competes with the high mobility group protein OsHMGB1 for binding to the rice calmodulin-like protein OsCML31 to enhance rice susceptibility to Meloidogyne graminicola.

The root-knot nematode Meloidogyne graminicola secretes effectors into rice tissues to modulate host immunity. Here, we characterised MgCRT1, a calreticulin protein of M. graminicola, and identified its target in the plant. In situ hybridisation showed MgCRT1 mRNA accumulating in the subventral oesophageal gland in J2 nematodes. Immunolocalization indicated MgCRT1 localises in the giant cells during parasitism. Host-induced gene silencing of MgCRT1 reduced the infection ability of M. graminicola, while over-expressing MgCRT1 enhanced rice susceptibility to M. graminicola. A yeast two-hybrid approach identified the calmodulin-like protein OsCML31 as an interactor of MgCRT1. OsCML31 interacts with the high mobility group protein OsHMGB1 which is a conserved DNA binding protein. Knockout of OsCML31 or overexpression of OsHMGB1 in rice results in enhanced susceptibility to M. graminicola. In contrast, overexpression of OsCML31 or knockout of OsHMGB1 in rice decreases susceptibility to M. graminicola. The GST-pulldown and luciferase complementation imaging assay showed that MgCRT1 decreases the interaction of OsCML31 and OsHMGB1 in a competitive manner. In conclusion, when M. graminicola infects rice and secretes MgCRT1 into rice, MgCRT1 interacts with OsCML31 and decreases the association of OsCML31 with OsHMGB1, resulting in the release of OsHMGB1 to enhance rice susceptibility.

Tobacco mosaic virus hijacks its coat protein-interacting protein IP-L to inhibit NbCML30, a calmodulin-like protein, to enhance its infection.

Calcium is an important plant immune signal that is essential for activating host resistance, but how RNA viruses manipulate calcium signals to promote their infections remains largely unknown. Here, we demonstrated that tobacco mosaic virus (TMV) coat protein (CP)-interacting protein L (IP-L) associates with calmodulin-like protein 30 (NbCML30) in the cytoplasm and nucleus, and can suppress its expression at the nucleic acid and protein levels. NbCML30, which lacks the EF-hand conserved domain and cannot bind to Ca2+ , was located in the cytoplasm and nucleus and was downregulated by TMV infection. NbCML30 silencing promoted TMV infection, while its overexpression inhibited TMV infection by activating Ca2+ -dependent oxidative stress in plants. NbCML30-mediated resistance to TMV mainly depends on IP-L regulation as the facilitation of TMV infection by silencing NbCML30 was canceled by co-silencing NbCML30 and IP-L. Overall, these findings indicate that in the absence of any reported silencing suppressor activity, TMV CP manipulates IP-L to inhibit NbCML30, influencing its Ca2+ -dependent role in the oxidative stress response. These results lay a theoretical foundation that will enable us to engineer tobacco (Nicotiana spp.) with improved TMV resistance in the future.

A novel Medicago truncatula calmodulin-like protein (MtCML42) regulates cold tolerance and flowering time.

Calmodulin-like proteins (CMLs) are one of the Ca2+ sensors in plants, but the functions of most CMLs remain unknown. The regulation of cold tolerance and flowering time by MtCML42 in Medicago truncatula and the underlying mechanisms were investigated using MtCML42-overexpressing plants and cml42 Medicago mutants with a Tnt1 retrotransposon insertion. Compared with the wild type (WT), MtCML42-overexpressing lines had increased cold tolerance, whereas cml42 mutants showed decreased cold tolerance. The impaired cold tolerance in cml42 could b complemented by MtCML42 expression. The transcript levels of MtCBF1, MtCBF4, MtCOR413, MtCAS15, MtLTI6A, MtGolS1 and MtGolS2 and the concentrations of raffinose and sucrose were increased in response to cold treatment, whereas higher levels were observed in MtCML42-overexpressing lines and lower levels were observed in cml42 mutants. In addition, early flowering with upregulated MtFTa1 and downregulated MtABI5 transcripts was observed in MtCML42-overexpressing lines, whereas delayed flowering with downregulated MtFTa1 and upregulated MtABI5 was observed in cml42. MtABI5 expression could complement the flowering phenotype in the Arabidopsis mutant abi5. Our results suggest that MtCML42 positively regulates MtCBF1 and MtCBF4 expression, which in turn upregulates the expression of some COR genes, MtGolS1 and MtGolS2, which leads to raffinose accumulation and increased cold tolerance. MtCML42 regulates flowering time through sequentially downregulating MtABI5 and upregulating MtFTa1 expression.

Fibrillarin RNA methylase is an interacting protein of Cryptosporidium parvum calmodulin-like protein (CpCML).

Cryptosporidium parvum is an obligate protozoan parasite invading epithelial cells of small intestine of human and animals, and causing diarrheal disease. In apicomplexan parasites, calcium signaling can regulate many essential biological processes such as invasion and migration. As the main intracellular receptor for calcium ions, calmodulins control the activities of hundreds of enzymes and proteins. Calmodulin-like protein (CML) is an important member of the calmodulin family and may play a key role in C. parvum, however, the actual situation is still not clear. The present study aimed to identify the parasite interaction partner proteins of C. parvum calmodulin-like protein (CpCML). By constructing the cpcml bait plasmid, 5 potential CpCML - interacting proteins in C. parvum oocyst were screened by yeast-two-hybrid system (Y2H). Bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) were performed as subsequent validations. Fibrillarin RNA methylase (FBL) was identified via this screening method as CpCML interacting protein in C. parvum. The identification of this interaction made it possible to get a further understanding of the function of CpCML and its contribution to the pathogenicity of C. parvum.

Isolation and identification of sporozoite membrane protein of Cryptosporidium parvum and evaluation of calmodulin-like protein immune protection.

Until now, no completely effective parasite-specific drugs or vaccines have been approved for the treatment of cryptosporidiosis. Through the separation and identification of the sporozoite membrane protein of Cryptosporidium parvum (C. parvum), 20 related proteins were obtained. Among them, a calmodulin-like protein (CML) has a similar functional domain-exchange factor hand (EF-hand) motif as calmodulin proteins (CaMs), so it may play a similarly important role in the invasion process. A 663 bp full gene encoding the C. parvum calmodulin-like protein (CpCML) was inserted in pET28a vector and expressed in Escherichia coli. An immunofluorescence assay showed that CpCML was mainly located on the surface of the sporozoites. Three-week-old female BALB/c mice were used for modelling the immunoreactions and immunoprotection of recombinant CpCML (rCpCML) against artificial Cryptosporidium tyzzeri infections. The results indicated a significantly increased in anti-CpCML antibody response, which was induced by the immunized recombinant protein. Compared to rP23 (recombinant P23), GST6P-1 (expressed by pGEX-6P-1 transfected E. coli), GST4T-1 (expressed by pGEX-4T-1 transfected E. coli), glutathione (GSH), adjuvant and blank control groups, rCpCML-immunized mice produced specific spleen cell proliferation in addition to different production levels of IL-2, IFN-γ, TNF-α, IL-4 and IL-5. Additionally, immunization with rCpCML led to 34.08% reduction of oocyst shedding in C. tyzzeri infected mice faeces which was similar to rP23. These results suggest that CpCML may be developed as a potential vaccine candidate antigen against cryptosporidiosis.

The small EF-hand protein CALML4 functions as a critical myosin light chain within the intermicrovillar adhesion complex.

Specialized transporting and sensory epithelial cells employ homologous protocadherin-based adhesion complexes to remodel their apical membrane protrusions into organized functional arrays. Within the intestine, the nutrient-transporting enterocytes utilize the intermicrovillar adhesion complex (IMAC) to assemble their apical microvilli into an ordered brush border. The IMAC bears remarkable homology to the Usher complex, whose disruption results in the sensory disorder type 1 Usher syndrome (USH1). However, the entire complement of proteins that comprise both the IMAC and Usher complex are not yet fully elucidated. Using a protein isolation strategy to recover the IMAC, we have identified the small EF-hand protein calmodulin-like protein 4 (CALML4) as an IMAC component. Consistent with this finding, we show that CALML4 exhibits marked enrichment at the distal tips of enterocyte microvilli, the site of IMAC function, and is a direct binding partner of the IMAC component myosin-7b. Moreover, distal tip enrichment of CALML4 is strictly dependent upon its association with myosin-7b, with CALML4 acting as a light chain for this myosin. We further show that genetic disruption of CALML4 within enterocytes results in brush border assembly defects that mirror the loss of other IMAC components and that CALML4 can also associate with the Usher complex component myosin-7a. Our study further defines the molecular composition and protein-protein interaction network of the IMAC and Usher complex and may also shed light on the etiology of the sensory disorder USH1H.

Transcriptome Analysis of Pear Leaves in Response to Calcium Treatment During Botryosphaeria dothidea Infection.

Pear (Pyrus bretschneideri), one of the most widely planted fruit trees in the world, is infected by pear ring rot disease, which is triggered by Botryosphaeria dothidea. Previous research has shown that exogenous calcium enhanced pear resistance to B. dothidea. To explore the molecular mechanism of calcium in pear pathogen resistance, we searched the differentially expressed genes (DEGs) between calcium and H2O treatment with B. dothidea inoculation in pear by using RNA-seq data. On the basis of the standard of a proportion of calcium/H2O fold change >2, and the false discovery rate (FDR) <0.05, 2,812 and 572 genes with significant differential expression were identified between the H2O and calcium treatments under B. dothidea inoculation at 2 days postinoculation (dpi) (D2) and 8 dpi (D8), respectively, indicating that significantly more genes in D2 responded to calcium treatment. Results of the gene annotation showed that DEGs were focused on plant-pathogen interactions, hormone signal transduction, and phenylpropanoid biosynthesis in D2. Moreover, transient silencing of PbrCML30 (pear calmodulin-like proteins 30), which had significantly higher expression in response to calcium than H2O treatments, conferred compromised resistance to B. dothidea. Exogenous calcium treatment slightly alleviated the symptoms of TRV2-PbrCML30 leaves compared with TRV2 leaves under inoculation, supporting its key role in pear resistance to B. dothidea. Overall, the information obtained in this study provides a possible mechanism of calcium in regulating pear resistance to B. dothidea.

Alfalfa (Medicago sativa L.) MsCML46 gene encoding calmodulin-like protein confers tolerance to abiotic stress in tobacco.

KEY MESSAGE: MsCML46 enhances tolerance to abiotic stresses through alleviating osmotic stress and oxidative damage by regulating the expression of stress-related genes to optimize osmolytes levels and antioxidant enzyme activity in transgenic tobacco. Abiotic stresses are major environmental factors that constraint crop productivity worldwide. Various stimuli regulate intracellular calcium levels and calcium-mediated signal transduction, and cellular responses. Ca2+ signals are perceived by different Ca2+ receptors. Calmodulin-like protein (CML) is one of the best-characterized Ca2+ sensors which shares sequence similarity with highly conserved calmodulin (CaM) ubiquitously expressed in plants. Currently, the molecular and physiological functions of CMLs are largely unknown. In this study, the MsCML46 was characterized in alfalfa (Medicago sativa cv. Zhaodong) under freezing stress. Results showed that MsCML46 was localized to the cytoplasm of Arabidopsis, and its expression was strongly elevated by cold, drought, salt, saline-alkali, and ABA treatments. Overexpressing MsCML46 in tobacco enhanced tolerance to freezing, drought, and salt stresses as evidenced by improved contents of osmotic regulatory solutes and antioxidant enzyme activity but decreased reactive oxygen species (ROS) accumulation. Furthermore, cold, drought, and salt stresses increased the expression of stress-related genes in transgenic tobacco. MsCML46 binds free Ca2+ to promote signal transduction and maintain higher K+/Na+ ratio. In this way, it protects intracellular homeostasis under sodium ion toxicity. These results suggest that MsCML46 plays a crucial role in resisting abiotic stresses and can be exploited in genetic engineering for crops.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana.

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp-CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein-peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

Characteristics of SlCML39, a Tomato Calmodulin-like Gene, and Its Negative Role in High Temperature Tolerance of Arabidopsis thaliana during Germination and Seedling Growth.

Calmodulin-like (CML) proteins are primary calcium sensors and function in plant growth and response to stress stimuli. However, so far, the function of plant CML proteins, including tomato, is still unclear. Previously, it was found that a tomato (Solanum lycopersicum) CML, here named SlCML39, was significantly induced by high temperature (HT) at transcription level, but its biological function is scarce. In this study, the characteristics of SlCML39 and its role in HT tolerance were studied. SlCML39 encodes a protein of 201 amino acids containing four EF hand motifs. Many cis-acting elements related to plant stress and hormone response appear in the promoter regions of SlCML39. SlCML39 is mainly expressed in the root, stem, and leaf and can be regulated by HT, cold, drought, and salt stresses as well as ABA and H2O2. Furthermore, heterologous overexpression of SlCML39 reduces HT tolerance in Arabidopsis thaliana at the germination and seedling growth stages. To better understand the molecular mechanism of SlCML39, the downstream gene network regulated by SlCML39 under HT was analyzed by RNA-Seq. Interestingly, we found that many genes involved in stress responses as well as ABA signal pathway are down-regulated in the transgenic seedlings under HT stress, such as KIN1, RD29B, RD26, and MAP3K18. Collectively, these data indicate that SlCML39 acts as an important negative regulator in response to HT stress, which might be mediated by the ABA signal pathway.

The Same against Many: AtCML8, a Ca2+ Sensor Acting as a Positive Regulator of Defense Responses against Several Plant Pathogens.

Calcium signals are crucial for the activation and coordination of signaling cascades leading to the establishment of plant defense mechanisms. Here, we studied the contribution of CML8, an Arabidopsis calmodulin-like protein in response to Ralstonia solanacearum and to pathogens with different lifestyles, such as Xanthomonas campestris pv. campestris and Phytophtora capsici. We used pathogenic infection assays, gene expression, RNA-seq approaches, and comparative analysis of public data on CML8 knockdown and overexpressing Arabidopsis lines to demonstrate that CML8 contributes to defense mechanisms against pathogenic bacteria and oomycetes. CML8 gene expression is finely regulated at the root level and manipulated during infection with Ralstonia, and CML8 overexpression confers better plant tolerance. To understand the processes controlled by CML8, genes differentially expressed at the root level in the first hours of infection have been identified. Overexpression of CML8 also confers better tolerance against Xanthomonas and Phytophtora, and most of the genes differentially expressed in response to Ralstonia are differentially expressed in these different pathosystems. Collectively, CML8 acts as a positive regulator against Ralstonia solanaceraum and against other vascular or root pathogens, suggesting that CML8 is a multifunctional protein that regulates common downstream processes involved in the defense response of plants to several pathogens.

Comparative transcriptome analysis to elucidate the enhanced thermotolerance of tea plants (Camellia sinensis) treated with exogenous calcium.

MAIN CONCLUSION: The molecular mechanisms regulating calcium-mediated thermotolerance in Camellia sinensis were revealed by RNA-Sequencing. Heat stress is one of the most remarkable abiotic factors limiting the growth and productivity of Camellia sinensis plants. Calcium helps regulate plant responses to various adverse environmental conditions, including heat stress. In this study, the effects of exogenous calcium on the physiological characteristics of heat-stressed C. sinensis were investigated. A calcium pretreatment increased the proline, soluble sugar, Ca2+, and chlorophyll contents, but decreased the malondialdehyde content and relative electrical conductivity in C. sinensis leaves under heat stress. Further analysis of the ultra-structure of chloroplasts indicated that heat stress induced accumulation of starch granules and destruction of the stroma lamella in C. sinensis. However, calcium pretreatment counteracted the adverse effects of heat stress on the structure of the photosynthetic apparatus. These results imply that the calcium pretreatment increased C. sinensis thermotolerance. Moreover, RNA-sequencing was applied to characterize the calcium-mediated transcript-level responses to heat stress. A total of 923 differentially expressed genes (DEGs) including 299 up-regulated and 624 down-regulated genes were identified. Functional annotations indicated that these DEGs were primarily related to signal transduction, transcriptional regulation, and post-translational modification. In addition, a C. sinensis gene [CsCML45 (GenBank: KY652927)] encoding a calmodulin-like protein was isolated. The heterologous expression of CsCML45 enhanced the thermotolerance of transgenic Arabidopsis thaliana plants. These results may be useful for characterizing the calcium-mediated molecular mechanism responsible for C. sinensis thermotolerance.

OsCML16 interacts with a novel CC-NBS-LRR protein OsPi304 in the Ca2+/Mg2+ dependent and independent manner in rice.

In plants, many target proteins of calmodulins (CaMs) have been identified in cellular metabolism and responses. However, calmodulin-like proteins (CMLs) and their target proteins have not been discovered in stress responses in rice. In this study, a novel CC-NBS-LRR protein was obtained in screening a cold stress rice seedlings yeast cDNA library with OsCML16 as bait. Furthermore, yeast two-hybrid and BiFC assays demonstrated that the full length, CC region in the N-terminus and LRR in the C-terminus of Pi304 protein could interact with OsCML16. More interestingly, OsCML16 bound to the 1-10 motif rather than 1-14 motif in the Ca2+ or Mg2+ dependent manner in vitro. In addition, transcript levels of OsCML16 and OsPi304 were induced more markedly in Nipponbare than in 9311 under cold stress. Taken together, these data indicates that they are involved in the cold stress signaling and response in rice.

rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming.

Primary infection of a plant with a pathogen that causes high accumulation of salicylic acid in the plant typically via a hypersensitive response confers enhanced resistance against secondary infection with a broad spectrum of pathogens, including viruses. This phenomenon is called systemic acquired resistance (SAR), which is a plant priming for adaption to repeated biotic stress. However, the molecular mechanisms of SAR-mediated enhanced inhibition, especially of virus infection, remain unclear. Here, we show that SAR against cucumber mosaic virus (CMV) in tobacco plants (Nicotiana tabacum) involves a calmodulin-like protein, rgs-CaM. We previously reported the antiviral function of rgs-CaM, which binds to and directs degradation of viral RNA silencing suppressors (RSSs), including CMV 2b, via autophagy. We found that rgs-CaM-mediated immunity is ineffective against CMV infection in normally growing tobacco plants but is activated as a result of SAR induction via salicylic acid signaling. We then analyzed the effect of overexpression of rgs-CaM on salicylic acid signaling. Overexpressed and ectopically expressed rgs-CaM induced defense reactions, including cell death, generation of reactive oxygen species, and salicylic acid signaling. Further analysis using a combination of the salicylic acid analogue benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and the Ca2+ ionophore A23187 revealed that rgs-CaM functions as an immune receptor that induces salicylic acid signaling by simultaneously perceiving both viral RSS and Ca2+ influx as infection cues, implying its autoactivation. Thus, secondary infection of SAR-induced tobacco plants with CMV seems to be effectively inhibited through 2b recognition and degradation by rgs-CaM, leading to reinforcement of antiviral RNA silencing and other salicylic acid-mediated antiviral responses.IMPORTANCE Even without an acquired immune system like that in vertebrates, plants show enhanced whole-plant resistance against secondary infection with pathogens; this so-called systemic acquired resistance (SAR) has been known for more than half a century and continues to be extensively studied. SAR-induced plants strongly and rapidly express a number of antibiotics and pathogenesis-related proteins targeted against secondary infection, which can account for enhanced resistance against bacterial and fungal pathogens but are not thought to control viral infection. This study showed that enhanced resistance against cucumber mosaic virus is caused by a tobacco calmodulin-like protein, rgs-CaM, which detects and counteracts the major viral virulence factor (RNA silencing suppressor) after SAR induction. rgs-CaM-mediated SAR illustrates the growth versus defense trade-off in plants, as it targets the major virulence factor only under specific biotic stress conditions, thus avoiding the cost of constitutive activation while reducing the damage from virus infection.

Towards Understanding Plant Calcium Signaling through Calmodulin-Like Proteins: A Biochemical and Structural Perspective.

Ca2+ ions play a key role in a wide variety of environmental responses and developmental processes in plants, and several protein families with Ca2+-binding domains have evolved to meet these needs, including calmodulin (CaM) and calmodulin-like proteins (CMLs). These proteins have no catalytic activity, but rather act as sensor relays that regulate downstream targets. While CaM is well-studied, CMLs remain poorly characterized at both the structural and functional levels, even if they are the largest class of Ca2+ sensors in plants. The major structural theme in CMLs consists of EF-hands, and variations in these domains are predicted to significantly contribute to the functional versatility of CMLs. Herein, we focus on recent advances in understanding the features of CMLs from biochemical and structural points of view. The analysis of the metal binding and structural properties of CMLs can provide valuable insight into how such a vast array of CML proteins can coexist, with no apparent functional redundancy, and how these proteins contribute to cellular signaling while maintaining properties that are distinct from CaM and other Ca2+ sensors. An overview of the principal techniques used to study the biochemical properties of these interesting Ca2+ sensors is also presented.

Overexpression of OsERF48 causes regulation of OsCML16, a calmodulin-like protein gene that enhances root growth and drought tolerance.

The AP2/ERF family is a plant-specific transcription factor family whose members have been associated with various developmental processes and stress tolerance. Here, we functionally characterized the drought-inducible OsERF48, a group Ib member of the rice ERF family with four conserved motifs, CMI-1, -2, -3 and -4. A transactivation assay in yeast revealed that the C-terminal CMI-1 motif was essential for OsERF48 transcriptional activity. When OsERF48 was overexpressed in an either a root-specific (ROXOsERF48 ) or whole-body (OXOsERF48 ) manner, transgenic plants showed a longer and denser root phenotype compared to the nontransgenic (NT) controls. When plants were grown on a 40% polyethylene glycol-infused medium under in vitro drought conditions, ROXOsERF48 plants showed a more vigorous root growth than OXOsERF48 and NT plants. In addition, the ROXOsERF48 plants exhibited higher grain yield than OXOsERF48 and NT plants under field-drought conditions. We constructed a putative OsERF48 regulatory network by cross-referencing ROXOsERF48 root-specific RNA-seq data with a co-expression network database, from which we inferred the involvement of 20 drought-related genes in OsERF48-mediated responses. These included genes annotated as being involved in stress signalling, carbohydrate metabolism, cell-wall proteins and drought responses. They included, OsCML16, a key gene in calcium signalling during abiotic stress, which was shown to be a direct target of OsERF48 by chromatin immunoprecipitation-qPCR analysis and a transient protoplast expression assay. Our results demonstrated that OsERF48 regulates OsCML16, a calmodulin-like protein gene that enhances root growth and drought tolerance.

First feed affects the expressions of microRNA and their targets in Atlantic cod.

To our knowledge, there is no report on microRNA (miRNA) expression and their target analysis in relation to the type of the first feed and its effect on the further growth of fish. Atlantic cod (Gadus morhua) larvae have better growth and development performance when fed natural zooplankton as a start-feed, as compared with those fed typical aquaculture start-feeds. In our experiment, two groups of Atlantic cod larvae were fed reference feed (zooplankton, mostly copepods, filtered from a seawater pond) v. aquaculture feeds: enriched rotifers (Brachionus sp.) and later brine shrimp (Artemia salina). We examined the miRNA expressions of six defined developmental stages as determined and standardised by body length from first feeding for both diet groups. We found eight miRNA (miR-9, miR-19a, miR-130b, miR-146, miR-181a, miR-192, miR-206 and miR-11240) differentially expressed between the two feeding groups in at least one developmental stage. We verified the next-generation sequencing data using real-time RT-PCR. We found 397 putative targets (mRNA) to the differentially expressed miRNA; eighteen of these mRNA showed differential expression in at least one stage. The patterns of differentially expressed miRNA and their putative target mRNA were mostly inverse, but sometimes also concurrent. The predicted miRNA targets were involved in different pathways, including metabolic, phototransduction and signalling pathways. The results of this study provide new nutrigenomic information on the potential role of miRNA in mediating nutritional effects on growth during the start-feeding period in fish larvae.