Cloning and pharmaceutical analysis of CaMK gene of Botrytis cinerea

Abstract

Using a PCR homology approach, DNA and cDNA sequences of calcium/calmodulin-dependent protein kinase (CaMK) gene of Botrytis cinerea were obtained. Southern blotting result displayed that CaMK was single copy in the genome of B. cinerea. The cDNA sequence of CaMK revealed an open reading frame of 2190 nucleotides encoding a 730 amino acid protein with predicted molecular weight of 81.8748 kDa. The genomic sequence of CaMK revealed the same ORF interrupted by six introns. Bioinformatics analysis showed that this protein had the distinctive features that characterize CaMK ATP binding region signature and serine/threonine protein kinase active-site signature. Pharmaceutical analysis displayed that the CaMK specific inhibitor, KN-62, could inhibit conidial germination, pathogenicity and herbicidal activity of B. cinerea BC4 strain. It was suggested that CaMK played an important role in regulating conidial germination, pathogenicity and herbicidal activity of B. cinerea.

Keywords

Botrytis cinerea / CaMK / pharmaceutical analysis / KN-62

Introduction

Botrytis cinerea is the causal agent of gray mold diseases on a wide variety of crop plants; at the same time, it can produce bioactive compounds when it is cultured under certain conditions (Chen et al., 2008). It was reported that mycotoxins from B. cinerea could be developed as microbial herbicides due to their high herbicidal bioactivity (Li, 2003; Zheng et al., 2008). Because chemical herbicides can cause pollution of environments, the use of microbial fungicides is a trend (Anand et al., 2010). For example, Trichoderma was used as commercial biofungicides for control of plant pathogens (Mukherjee and Kenerley, 2010). Using B. cinerea BC4 and its mutant isolates (strong and weak toxic isolates) as materials, herbicidal substances and gene differential expression in B. cinerea were studied by Ma (2006). Through the method of DDRT-PCR (differential display reverse transcription-PCR), a herbicidal activity-related fragment was obtained. Sequence analysis showed that it had high similarity to calcium/calmodulin-dependent protein kinase. However, the toxigenic heredity mechanism of B. cinerea is not clear at present. Based on known CaMK genes in GenBank database, DNA and cDNA sequences of CaMK of B. cinerea BC4 were cloned by using PCR homology approach in this study. Pharmaceutical analysis for the function of CaMK was performed to determine the effects of KN-62, a specific inhibitor of CaMK (Tokumitsu et al., 1990), on conidial germination, pathogenicity and herbicidal activity of the toxin of B. cinerea BC4. Results obtained would pave the way to study the regulating mechanism of CaMK gene in B. cinerea BC4 in this work.

Materials and methods

Fungal growth conditions

The isolates of B. cinerea strain BC4 used in this study corresponded to the collection from the Molecular Plant Pathology Laboratory, Agricultural University of Hebei, China, which were obtained from infected fruit tissue of tomato. B. cinerea was grown on 2% PDA plates for 10 d at 20°C. Spores of B. cinerea were resuspended in sterile water.

Cloning of CaMK in B. cinerea BC4

An appropriate method to extract DNA of CTAB was changed based on the description of Drenth et al. (1993). Total RNA was isolated with Trizol kit (TIANGEN) and then used to synthesize single-stranded cDNA. Using DNA and cDNA as template, respectively, CaMK gene was obtained by combining DC-F with DC-R primers. The sequences of DC-F and DC-R primers designed based on the CDS sequence of B. cinerea in GenBank were as follows: DC-F 5΄-ATGTCTTCATTCGCTGGCG-3΄ and DC-R 5΄-CTACTTCTCAGAGGTAGCCCCTTC-3΄. PCR reactions were carried out using 20 ng/µL DNA/cDNA, 1 × PCR buffer, 2.5 mmol/L dNTPs, 10 µmol/L primer each and 0.5 U Taq DNA polymerase in a total volume of 25 µL. PCR amplification was performed for 31 cycles (98°C for 10 s, 56°C for 30 s, 68°C for 2.5 min) followed by final extension for 10 min at 72°C. The PCR products were resolved on 1.2% TAE-agarose gel and purified from an agarose gel slice using the UNIQ-10 Gel Extraction Kit (Sangon). The purified PCR products were cloned into the plasmid pMD-19 simple vector (Takara) following the manufacturer’s instructions. M13 primers were used to generate single pass partial sequences of the plasmid with the differential band inserts. The positive clones were sequenced using an automated sequencer (ABI PRISM 3730XL, Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.).

Bioinformatics analysis

The cDNA and DNA sequences of CaMK were compared by DNAMAN software. The CaMK protein character was predicted by DNAStar. The conserved domain was analyzed by ScanPro site. The secondary structure of CaMK protein was analyzed by SOPMA, and the three-dimensional mode was constructed by SWISS-MODEL.

Southern blotting

Genomic DNA of B. cinerea strain BC4 was digested with EcoRV that was cut at a single site within the probe-specific CaMK gene sequence and with EcoRI, HindIII and KpnI restriction enzymes that couldn’t cut the probe-specific sequence. The digested DNA was electrophoresised on 0.8% TAE-agarose gel and transferred to a nylon membrane (AMRESCO). Hybridization, washes and hybrid detection were done according to the instructions provided with DIG DNA labeling and detection kit (Roche).

Assay of conidial germination, pathogenicity, herbicidal activity of the toxin extracted from B. cinerea BC4

KN-62 (purchased from ALEXIS Corporation), a specific inhibitor of CaMK, was used to determine the effects of CaMK on conidial germination, pathogenicity and herbicidal activity of the toxin of B. cinerea BC4. For the inhibition of pharmaceutical biosynthesis, spores of B. cinerea BC4 were suspended in KN-62 at the concentrations of 20 µmol/L, 40µmol/L, 60 µmol/L, 80 µmol/L and 100 µmol/L, respectively, and then 50 µL conidial suspension (2 × 10³ speres/mL) was titrated on the plastic film and cultured in Petri dishes containing moist bibulous paper for 11 h in the dark at 20°C. At the same time, sterilized ddH₂O was used as control, and the experiment was repeated at least three times with duplicates in each experiment. Conidial germination was observed using a microscope.

B. cinerea BC4 inoculated in PDA media (Φ = 90 mm) containing 80 µmol/L KN-62 and cultured in darkness at 20°C, with sterile water used as control, was inoculated for 10 d, and mature tomatoes were infected by placing a piece of agar of 0.8 cm diameter, containing B. cinerea mycelium. The lesion diameter of tomato was measured 2 days after inoculation to determine the statistical significance of the data. The experiment was repeated three times. Error bars were calculated as standard error.

PD liquid media were added with KN-62 at the final concentrations of 20 µmol/L, 40 µmol/L, 60 µmol/L, 80 µmol/L and 100 µmol/L, respectively, inoculated with mycelium plugs of B. cinerea BC4 (Φ = 6 mm) and cultured in darkness at 20°C for 21 d, with the toxin extracted, respectively. Stem-leaf dispose was used in this experiment. Digitaria sanguinalis was cultured at 25°C with 3800 lx of light and 70% RH. The toxin was sprayed on the weed using a sprayer till all the leaves were wet. Each treatment was repeated three times. After 12 h, weed growth was measured.

Results

Cloning of CaMK

Using single-stranded cDNA as template, cDNA sequence of CaMK was amplificated by DC-F/R primers. A special fragment about 2.1 kb was generated from B. cinerea BC4 (Fig. 1A). The special fragment was resolved, cloned and sequenced. A length of 2190 bp sequence was obtained and identified as the full-length cDNA complete sequence of CaMK. BLAST analysis result showed that it had 98% similarity to CaMK gene of B. cinerea. Using genomic DNA of B. cinerea BC4 as template, a special fragment about 2.5 kb was generated by DC-F/R primers (Fig. 1B). Cloning, sequencing and homologous analysis for the fragment indicated that the full-length DNA complete sequence of CaMK was 2540 bp.

Bioinformatics analysis

The bioinformatics character of CaMK gene was analyzed, indicating that 7 extrons and 6 introns existed in DNA of CaMK. The CaMK protein character predicted by DNAStar showed that the molecular weight and isoelectric point of the predicted protein was 81.8748 kDa and 6.22, respectively, with 730 amino acid residues including 95 alkaline amino acids (K, R), 116 acidic amino acids (D, E), 216 hydrophobous amino acids (AILFWV), and 160 polar amino acids (NCQSTY). The conserved domain was analyzed by ScanPro site, which indicated that the primary structure of CaMK contained ATP binding region signature and serine/threonine protein kinases active-site signature (Fig. 2).

CaMK protein had 51% homology to proteins containing the CaMK protein of Setosphaeria turcica by BLAST in NCBI results. The secondary structure of CaMK protein was analyzed by SOPMA (Fig. 3), which showed CaMK protein contained 37.26% alpha helix, 10.82% extend stand, 4.38% beta turn and 47.53% random coil.

Southern blotting

Southern blotting analysis was performed to determine the copy number of CaMK in the genome of B. cinerea BC4. Treatments with enzymes (EcoRI, HindIII and KpnI) that were not cut within the sequence consistent with the probe produced single bands, and two bands were observed after the treatment with EcoRV that was cut at a single position within the probe sequence (Fig. 4). These results indicated that CaMK was single copy in the genome of B. cinerea BC4.

KN-62 inhibited conidial germination of B. cinerea BC4

CaMK inhibitor KN-62 was used to treat conidia of B. cinerea BC4. An inhibitory effect of KN-62 was positively related to concentrations of KN-62. Approximately 50% spore could not germinate at 60 µmol/L concentration of KN-62 (Fig. 5A). With the increasing concentration of KN-62, the inhibitory effect was enhanced gradually. The inhibition rate of conidial germination at 80 µmol/L KN-62 was approximately 100% (Fig. 5B). These results indicated that KN-62 was able to inhibit conidial germination.

KN-62 inhibited pathogenicity of B. cinerea BC4

To analyze the effect of CaMK on virulence, conidial suspension of B. cinerea BC4 treated with CaMK inhibitor KN-62 was inoculated on the acupuncture surface of tomato. Lesion sizes were determined 3 days post inoculation (dpi). In the wild-type control, water-soaked lesions with a diameter of approximately 3 cm were formed 3 dpi. The B. cinerea treated with 80 µmol/L KN-62 was able to infect tomato, but it was strongly delayed in primary lesion formation and lesion diameters were smaller at 3 dpi compared with the wild type (Fig. 6). These results indicated that KN-62 was able to inhibit pathogenicity of B. cinerea.

KN-62 inhibited herbicidal activity of the toxin of B. cinerea BC4

Different toxins of B. cinerea BC4 treated with various KN-62 concentrations were isolated and sprayed on the D. sanguinalis. The result indicated that the herbicidal activity of toxins from B. cinerea treated with KN-62 was reduced, especially the herbicidal activity of B. cinerea treated with 80 µmol/L KN-62 was remarkably weakened (Fig. 7), indicating that KN-62 was able to inhibit herbicidal activity of the toxin of B. cinerea BC4.

Discussion

Calcium/calmodulin-dependent protein kinase (CaMK) is a multi-functional serine/threonine protein kinase, and it is a major member of Ca²⁺/CaM-regulated protein family. The multifunctional CaMK family includes CaM kinases I, II, III and V on the basis of different arrangement of the catalytic, regulatory and associated domains, whose activity is highly dependent on Ca²⁺/ calmodulin (Hanks and Quinn, 1991). In this study, we cloned a CaMK gene in B. cinerea, and the effects of inhibitors of the encoded protein on the conidial germination, pathogenicity and toxin activity were analyzed. CaMKs are key components in many calcium/CaM mediated pathways in animals and yeast (Ma et al., 2004; Timmins etal., 2009). CaMK function is various in different fungi. In the filamentous fungus Aspergillus nidulans, calcium/calmodulin-dependent protein kinases, CMKB was expressed on the nuclear division and affected conidia germination (Joseph and Means, 2000). The disruption of calcium/calmodulin-dependent protein kinases, CMKA and CMKB in filamentous fungus A. nidulans was reported to be lethal (Joseph and Means, 2002). In another filamentous fungus, in Sporothrix schenckii, SSCMKI inhibited effective mycelium transition (Valle-Aviles et al., 2007). The MoCMK1 gene encoding a putative Ca²⁺/calmodulin-dependent kinase plays key roles in the pathogenicity of the rice blast fungus (Liu et al., 2010). In Neurospora crassa, a Ca²⁺/calmodulin-dependent protein kinase regulated aflatoxin production and suppressed growth of CaMK-1 null strains (Jayashree et al., 2000; Yang et al., 2001). In Coprinus cinereus, a Ca²⁺/calmodulin-dependent protein kinase played crucial roles in mycelial growth (Kameshita et al., 2007). CaMK protein of B. cinerea was 71% similar to Ca²⁺/calmodulin dependent protein kinase A of A. nidulans and 53% similar to CaMK-II of Saccharomyces cerevisiae. Tsai et al. (2002) described a Ca²⁺/calmodulin-dependent protein kinase gene in the filamentous fungus, Arthrobotrys dactyloides, which encoded a protein with significant homology to mammalian CaMKs. Our study is the first report about cloning of CaMK gene in B. cinerea. Analysis result showed that conserved domain of Ca²⁺/calmodulin-dependent protein kinase such as ATP binding region signature and serine/threonine protein kinases active-site signature existed in CaMK of B. cinerea BC4. Southern blotting indicated that CaMK was single copy in the genome of B. cinerea BC4. To further elucidate the physiologic role of CaMK in B. cinerea, we examined the effect of KN-62, a membrane-permeable CaM kinase inhibitor (Praskova et al., 2002). After treatment by KN-62, conidial germination, pathogenicity and herbicidal active substances of B. cinerea were affected. Therefore, CaMK played an important regulation role in B. cinerea. The molecular mechanism of CaMK in conidial germination, pathogenicity and herbicidal activity of B. cinerea should be identified in the future. Using B. cinerea CaMK gene deletion mutants obtained by building the gene homologous recombination vector and transformation of B. cinerea protoplast, regulation mechanism of CaMK could be identified. Results about regulation mechanism of CaMK in B. cinerea can provide theoretical basis for controlling gray mold and developing new herbicides, etc.

References

[1] Anand T, Chandrasekaran A, Kuttalam S, Senthilraja G, Samiyappan R (2010). Integrated control of fruit rot and powdery mildew of chili using the biocontrol agent Pseudomonas fluorescens and a chemical fungicide. Biol Control, 52(1): 1–7

[2] Chen H, Xiao X, Wang J, Wu L J, Zheng Z M, Yu Z L (2008). Antagonistic effects of volatiles generated by Bacillus subtilis on spore germination and hyphal growth of the plant pathogen, Botrytis cinerea. Biotechnol Lett, 30(5): 919–923

[3] Drenth A, Goodwin S B, Fry W E, Davidse L C (1993). Genotypic diversity of Phytophthora infestans in the Netherlands revealed by DNA polymorphisms. Phytopathology, 83(10):1087–1092

[4] Hanks S K, Quinn A M (1991). Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol, 200: 38–62

[5] Jayashree T, Praveen Rao J, Subramanyam C (2000). Regulation of aflatoxin production by Ca²⁺/calmodulin-dependent protein phosphorylation and dephosphorylation. FEMS Microbiol Lett, 183(2): 215–219

[6] Joseph J D, Means A R (2000). Identification and characterization of two Ca²⁺/CaM-dependent protein kinases required for normal nuclear division in Aspergillus nidulans. J Biol Chem, 275(49): 38230–38238

[7] Joseph J D, Means A R (2002). Calcium binding is required for calmodulin function in Aspergillus nidulans. Eukaryot Cell, 1(1): 119–125

[8] Kameshita I, Yamada Y, Nishida T, Sugiyama Y, Sueyoshi N, Watanabe A, Asada Y (2007). Involvement of Ca²⁺/calmodulin-dependent protein kinases in mycelial growth of the basidiomycetous mushroom, Coprinus cinereus. Biochim Biophys Acta, 1770(9): 1395–1403

[9] Li C G (2003). Studies on herbicidal activity of metabolites from Botrytis cinerea and isolation of active substance. Dissertation for the Master Degree. Hebei: Agricultural University of Hebei (in Chinese)

[10] Liu X H, Lu J P, Dong B, Gu Y, Lin F C (2010). Disruption of MoCMK1, encoding a putative calcium/calmodulin-dependent kinase, in Magnaporthe oryzae. Microbiol Res, 165(5): 402–410

[11] Ma J (2006). Mutation and differential display of herbicidal-related genes of Botrytis Cinerea. Dissertation for the Master Degree. Hebei: Agricultural University of Hebei (in Chinese)

[12] Ma L, Liang S P, Jones R L, Lu Y T (2004). Characterization of a novel calcium/calmodulin-dependent protein kinase from tobacco. Plant Physiol, 135(3): 1280–1293

[13] Mukherjee P K, Kenerley C M (2010). Regulation of morphogenesis and biocontrol properties in Trichoderma virens by a VELVET protein, Vel1. Appl Environ Microbiol, 76(7): 2345–2352

[14] Praskova M, Kalenderova S, Miteva L, Poumay Y, Mitev V (2002). Ca²⁺ /calmodulin-dependent protein kinase (CaM-kinase) inhibitor KN-62 suppresses the activity of mitogen-activated protein kinase (MAPK), c-myc activation and human keratinocyte proliferation. Arch Dermatol Res, 294(4): 198–202

[15] Timmins J M, Ozcan L, Seimon T A, Li G, Malagelada C, Backs J, Backs T, Bassel-Duby R, Olson E N, Anderson M E, Tabas I (2009). Calcium/calmodulin-dependent protein kinase II links ER stress with Fas and mitochondrial apoptosis pathways. J Clin Invest, 119(10): 2925–2941

[16] Tokumitsu H, Chijiwa T, Hagiwara M, Mizutani A, Terasawa M, Hidaka H (1990). KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, a specific inhibitor of Ca²⁺ /calmodulin-dependent protein kinase II. J Biol Chem, 265(8): 4315–4320

[17] Tsai P J, Tu J, Chen T H (2002). Cloning of a Ca^2+€€/calmodulin-dependent protein kinase gene from the filamentous fungus Arthrobotrys dactyloides. FEMS Microbiol Lett, 212(1): 7–13

[18] Valle-Aviles L, Valentin-Berrios S, Gonzalez-Mendez R R, Rodriguez-Del Valle N (2007). Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii. BMC Microbiol, 7(1): 107

[19] Yang Y, Cheng P, Zhi G, Liu Y (2001). Identification of a calcium/calmodulin-dependent protein kinase that phosphorylates the Neurospora circadian clock protein FREQUENCY. J Biol Chem, 276(44): 41064–41072

[20] Zheng M, Xu K, Dong J G (2008). Purification and structural identification of herbicides from Botrytis cinerea. Acta Microbiologica Sinica, 48(10): 1362–1366 (in Chinese)

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg