Cloning And Functional Analysis Of CaM Genes Regulating The Growth, Development And Pathogenicity Of Setosphaeria Turcica

Northern Corn Leaf Blight, caused by Setosphaeria turcica, is one of the most important diseases in corn planting area.Therefore,it is important to acquire a detailed molecular understanding of the mechanisms of this fungal phytopathogenicity to effectively control it.Durintg infection and disease process,fungi,including S. turcica are subjected to recognizing susceptible host.The studying of interaction between maize and the fungus in the signaling pathways will lay foudation for following,such as the designing of new fungicide,putting forward new disease control measure,understanding of mechanisms of fungal phytopathogenicity.The paper mainly discuss the calcium signaling which control the conidium germination ,appressorium formation and pathogenicity of fungal pathogens.To elucidate if calcium signaling pathway is correlated with conidium germination and appressorium formation induced by hydrophobic surface in Setosphaeria turcica,conidia were treated by two kind of inhibitors.The TFP and CsA could inhibit germination and appressorium formation. This inhibtion was positively related to TFP and CsA concention.Under the same concentration of inhibitors , appressorium formation was inhibited more strongly than conidium germination.cDNA homologous fragments of the calmodulin gene were obtained by polymerase chain reaction (PCR) amplification from degenerated primer sets designed on the basis of the conserved amino acid regions of CaM domains from others fungis. The completed cDNA sequence of CaM in S. turcica was obtained by the method of SMART-RACE. There was 95%～100% identity of amino acid sequence with other fungal CaM gene including Cochliobolus miyabenus, Phaeosphaeria nodorum , Magnaporthe grisea. The CaM gene(EF010936) included a 776 bp DNA sequence with a 450 bp coding region,five exons and four introns. The predicated protein of CaM gene had 149 aa. All introns were accordance with GT-AG rules.The promoter of CaM was isolated with genomic walking strategy.Analysis of CaM promoter using the Proscan software showed that it contained several regulatory elements,such as TATA box,Sp1,AP2 and TFIID. The result of Southern-blot showed that CaM was single copy in the genome of S. turcica. To study what function CaM gene played in growth and pathogenesis of S. turcica, CaM antisense expressing vectors had been constructed and identified.The transformation system was established on the basis of screening marker of hygromycin B resistance and PEG-induced fusion of protoplasts and CaM antisense plasmid.Fourteen transformants were obtained and could all be subcultured in the PDA medium containing hygromycin B continuously.The transformants were identified by PCR and southern-bloting and two CaM antisense transformants were obtained.Under quinic acid induction ,the conidium germination decreased.A majority of the germinating conidia of CaM antisense transformant formed abnormal appressoria which accumulated less pigment and a part of conidia could not form appressoria. The CaM antisense mutant significantly reduced HT-toxin activity.Four calmodulin dependent protein kinases and calcineurin were cloned with the candidate gene cloning strategy.Calcineurin A encoded 525 aa .There was 86%～94% identity of amino acid sequence with other fungal Calcineurin A gene including P. nodorum, Sclerotinia sclerotiorum, Botrytis cinerea. The Calcineurin A gene(EF407562) included six introns and the intron were 116bp,47,47,47,47and 49 bp long respectively.Four genes encoding different calmodulin-dependent protein kinase have been characterized in the maize phytopathogenic fungus S. turcica.The kinases were cloned from S. turcica by RACE strategy. Three different calmodulin-dependent protein kinase were cloned with complete open reading frame and the others was partial .All the calmodulin-dependent protein kinase had the ATP-binding region signature and Serine/Threonine protein kinases active-site signature.The above results can summarize the function of CaM: insignificant effect on mycelium development and growth but related to the pigment accumulation in hypha; key factor regulating the conidial development; related to the HT-toxin activity.The cloning of calcineurin A and calmodulin-dependent protein kinase genes will lay foundation for undersanding of calcium signaling in the S. turcica.