Citrus Blue And Green Molds CaMK1 Gene Cloning And Function Analysis

Blue and green molds caused by Penicillium italicum and P.digitatum are the most serious fungal diseases to citrus fruits during postharvest storing and transportation.The sterol demethylation inhibitor fungicides(DMIs)can control these fungal diseases but produce noticeable DMIs-resistance problems.Thus researches on DMIs-resistance mechanisms are coming into scientific hotspots.Calcium(Ca2+)acting as a cellular second messenger can combine with calmodulin to initiate Ca2+-signal transduction,regulating fungal growth and development,morphogenesis,environmental adaptation,and fungicide resistance.Ca2+/calmodulin-dependent kinase(CaMK)acts as a crucial protein element in Ca2+-signal transduction pathways.However,the gene structure and function of such protein kinase in citrus molds remain obscure.Making comparative study on P.italicum and P.digitatum CaMK genes to reveal their function(s)is of great benefits to more insight into molecular mechanisms underlying Ca2+-signal regulation on citrus pathogens growth and development,environmental adaptation and fungicide resistance.The present study cloned CaMK genes from P.italicum and P.digitatum(PiCaMK1 and PdCaMK1,respectively),and investigated their functions in regulating citrus pathogens growth and development,virulence,environmental stimuli responses and DMIs resistance by constructing CaMK11-deleted and-complemented strains and subsequent phenotype analysis.The main results are listed below.1.The present study cloned PiCaMK1 according to transcriptome-originated P.italicum CaMK unigene sequence.The genomic DNA(gDNA)and cDNA of PiCaMK1 are 1379 bp and 1209 bp in full-length,respectively,comprising 3 introns and encoding 402 amino-acid residues.The present study also cloned PdCaMKl according to homolog gene sequence from PiCaMK1-originated BLAST.The gDNA and cDNA of PdCaMK1 are 1373 bp and 1221 bp in full-length,respectively,comprising 3 introns and encoding 406 amino-acid residues.The two CaMK1s exhibited>85%sequence similarity,both containing conserved serine/threonine protein kinase catalytic domain.The present study constructs P.italicum and P.digitatum CaMK1-deleted strains according to homologous recombination gene-knockout strategy and constructs P.italicum CaMK1-complemented strain by Agrobacterium-mediated fungal-transformation.2.The present study compared the effects of CoMK1-deletion on the two citrus pathogens vegetative growth.P.italicum and P.digitatum CaMK1-deleted strains both showed slower vegetative growth.After culturing on PDA plate for 7 days,The colony diameter(25.5 mm)of P.italicum CaMK1-deleted strain was 52.3%decreased in comparison with wild-type strain(53.5 mm)with mycelial morphogenesis changed.The vegetative growth of CaMK1-complemented P.italicum strain was recovered to the level of wild-type strain.The colony diameter(25.7 mm)of P.digitatum CaMK1-deleted strain was 42.5%decreased in comparison with wild-type strain(44.7 mm).The results indicated the involvement of CaMKl in regulating two citrus pathogens vegetative growth with similar effects.3.The present study compared the effects of CaMK1-deletion on the two citrus pathogens sporulation and virulence.P.italicum and P.digitatum CaMK1-deleted strains showed 65.9%and 64.2%decrease in sporulation,respectively,as compared with their corresponding wild-type strains.CaMK1-deleted strains from the two citrus pathogens both exhibited slower spreading rates on citrus epidermis,i.e.,showing 66.8%and 67.5%decrease in virulence,respectively,as compared with corresponding wild-type strains.The results indicated the involvement of CaMK1 in regulating two citrus pathogens sporulation and virulence with similar effects.4.The present study compared the effects of CaMK1-deletion on the two citrus pathogens DMIs resistance.The deletion of PiCaMK1 has no significant effect on P.italicum DMIs-resistance.In contrast,the deletion of PdCaMK1 significantly reduced P.digitatum DMIs-resistance.The prochloraz and imazalil EC50 values of PdCaMKl-deleted strain were respectively decreased to 55.3%and 61.9%of wild-type level.The results indicated differential effects of CaMK1 in regulating the two citrus pathogens DMIs-resistance.5.The present study compared the effects of CaMK1-deletion on the two citrus pathogens responses to environmental stimuli including stress condition(s).Salt stress(e.g.,1.2 mol·L-1 NaCl or KC1 treatment)inhibited P.italicum wild-type strain in vegetative growth by 15.2%and 20.8%,respectively,and inhibited P.italicum CaMK1-deleted strain in vegetative growth by 4.3%and 7.4%,respectively,indicating that the CaMK1-deletion improved P.italicum tolerance to salt stress.The same salt stress condition also inhibited P.digitatum wild-type and CaMK1-deleted strains in vegetative growth,and the inhibiting rate was higher in CaMK1-deleted strain,indicating PdCaMK1-deletion further reduced P.digitatum tolerance to salt stress.Osmotic regulator mannitol at 0.6 or 0.9 mol·L-1 accelerated vegetative growth for P.italicum wild-type and PiCaMK1-deleted strains with higher accelerating-rate in wild-type strain,but had no significant effects on P.digitatum vegetative growth concerning both wild-type and CaMK1-deleted strains.Oxidative stress H2O2 at 6.0 or 8.0 mmol·L-1 accelerated vegetative growth for P.digitatum wild-type and PdCaMK1-deleted strains with lower accelerating-rate in wild-type strain,but had no significant effects on P.italicum vegetative growth concerning both wild-type and CaMK1-deleted strains.The results indicated the involvement of CaMKl in regulating two citrus pathogens responses to environmental stimuli with different mechanisms.