Identification Of Polyketide Synthase And Effector In Nimbya Alternantherae Based On Genome Analysis

Alligatorweed(Alternanthera philoxeroides)is native to South American,but has become a very aggressive invader in many regions of the world.This weed has strong adaptability and spreads fast,which results in heavy impact on agriculture and ecology.Traditional control methods are not effective for this weed,and may themselves cause environment pollution.Therefore many researchers nationally and overseas have been investigating safe and efficient methods of biocontrol.Nimbya alternantherae is considered a potential biocontrol fungus.This organism produces pathotoxins,one of which is known as vulculic acid,a polyketide,and causes the disease of alligatorweed.This study focused on the prediction and analysis of polyketide synthase(PKS)and effectors based on complete genome sequencing and transcriptome sequencing(RNA-seq)of the fungus.The major results of the study are presented below.Illumina/Solexa sequencing with the HiSeq2000 platform,and assembly and gene prediction with different software programs revealed that isolate NA2 of N.altheranhera has an assembled genome size of 34 MB with 49% GC content and 10470 predicted genes.After analysis with the program HMMER,12 putative fungal typeⅠPKS genes were found,11 of which showed strong homology with the PKS genes of other ascomycetous fungi by BLAST against the NCBI NR database and after GO annotation.Predicted genes were filtered by length(<300 amino acid),presence of transdomain signal peptide,and occurrence of conserved motif RxLx or Y/W/FxC,to reveal 54 putative effectors.Among these,6 had no matches in the NR database,43 were hypothetical proteins without specific functions and 5 were identified with a specific putative function.To examine gene expression,alligatorweed leaves were inoculated with N.altheranhera,and RNA-seq samples were generated at various times over 72 hours;in addition two RNA-seq samples from mycelial shake cultures,one from a toxin-producing medium(modified Fries3)and one from a non-toxin-producing medium(potato carrot),were produced.These samples were also subjected to high throughput sequencing on a HiSeq2000 platform.Through BLAT alignment and cluster analysis,elongation factor 2(EF2)was confirmed as reference genes for relative gene expression analysis.A normalized value,FPKM,was calculated for each transcript expression,through FPKM clustering,8 putative PKS genes were found to have relatively high expression among 12 detected in inoculated alligatorweed leaves;among the hightly-expressed putative PKS genes,3 had relatively high expression in the mycelia from Fries3 than that from PC.17 of 54 putative effectors had relatively high expression in inoculated alligatorweed leaves,with a variety of expression patterns,among the hightly-expressed putative effector genes,13 had relatively high expression in the mycelia from Fries3 than that from PC.