Identification and characterization of LysM effectors in Penicillium expansum

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Abstract

P. expansum is regarded as one of the most important postharvest rots of apple fruit and is also of great concern to fruit processing industries. Elucidating the pathogenicity mechanism of this pathogen is of utmost importance for the development of effective and safe management strategies. Although, many studies on modification of the host environment by the pathogen were done, its interactions with fruit during the early stages of infection and the virulence factors that mediate pathogenicity have not been fully defined. Effectors carrying LysM domain have been identified in numerous pathogenic fungi and their role in the first stages of infection has been established. In this study, we identified 18 LysM genes in the P. expansum genome. Amino acid sequence analysis indicated that P. expansum LysM proteins belong to a clade of fungal-specific LysM. Eleven of the discovered LysM genes were found to have secretory pathway signal peptide, among them, 4 (PeLysM1 PeLysM2, PeLysM3 and PeLysM4) were found to be highly expressed during the infection and development of decay of apple fruit. Effect of targeted deletion of the four putative PeLysM effectors on the growth and pathogenicity was studied. Possible interactions of PeLysM with host proteins was investigated using the yeast-two-hybrid system.

Figures

  • Table 1. Genes containing LysM with predicted signal peptide found in P.expansum genome.
  • Table 2. Genes containing LysM without signal peptide found in P.expansum genome.
  • Fig 1. Analysis of LysM domains found in P. expansum genome. (A) Multiple alignment of all predicted LysM domains found in the genome of P.expansum. (B) Sequence logo for the multiple alignment. (C) Sequence logo for LysM domain defined by Protein A of Staphylococcus aureus (Prosite PS51782). (D) Sequence logo for consensus of fungal-specific LysM motifs [21].
  • Fig 2. Expression of secreted LysM genes based on the analysis of the RNAseq data of apples infected with PEX1. The RNAseq data was taken from Ballester et al. [41]. RNA-Seq data were generated from four different libraries: a cDNA library synthesized from a mixture of RNAs obtained from P. expansum PEX1 spores collected after 7 days of growth in PDA and from healthy, non-inoculated fruits, and three libraries from P. expansum–infected apples at 24, 48, and 72 hpi, respectively. The relative expression of the genes is expressed as RPKM—Reads Per Kilobase of transcript per Million mapped reads.
  • Fig 3. Expression of the PeLysM1, PeLysM2, PeLysM3 and PeLysM4 in PDB culture and during infection and development of Penicillium expansum on apple fruit. Relative gene expression was calculated from Cq values using a ΔΔCq method [40]. The results are an average of three independent measurements. Bars indicate standard error.
  • Fig 4. Gene structure and phylogenetic analysis of the four putative PeLysM effectors. (A) Diagrammatic representation of the structures of the four PeLysM genes. Black bars indicate the predicted exons present in the gene model, green rectangles- LysM domains, blue rectangle—lysozyme domain. The genomic sequence length in bp is given for each gene as an indication of scale; (B) Phylogram with distance indicator showing the relatedness of the LysM domains from the four P. expansum putative effectors to known fungal effector-proteins based on the alignment of individual LysM domains from these proteins. Each color indicates different specie: blue–P. expansum, purple–T. atroviride, red–M. graminicola, green–C. fulvum, yellow–M. oryzae, brown–V. dahlia, pink–C. higginsianum.
  • Fig 5. Effect of the targeted deletion on the growth rate and colony morphology. (A) Colony morphology at 7 dpi (B) radial growth on PDA medium of the wild type (P. e 100), ectopic mutants (E), and knockout mutants for PeLysM1 (Δ1), PeLysM2 (Δ2), PeLysM3(Δ3), and PeLysM4(Δ4). Radial growth results are an average of three independent measurements. Bars indicate standard error Statistical analysis present in S2 Table.
  • Fig 6. Effect of the target deletion on the germination. (A) Percent of germinating spores, (B) length of the germ tubes and (C) microscope observation of the germination of spore 18 hr post inoculation on 1.5% agar plates. Wild type (P. e 100), ectopic mutants (E), knockout mutants for PeLysM1 (Δ1), PeLysM2 (Δ2), PeLysM3(Δ3), and PeLysM4(Δ4). Percent of germination is an average of three different microscopic fields including 100–120 spores each. Length of the germs is an average of more than 50 measurements. Bars indicate standard error. Different letters indicate significant differences at P<0.05 based on nested one-way ANOVA followed by Tukey’s honest significant difference (HSD) test.

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Levin, E., Ballester, A. R., Raphael, G., Feigenberg, O., Liu, Y., Norelli, J., … Droby, S. (2017). Identification and characterization of LysM effectors in Penicillium expansum. PLoS ONE, 12(10). https://doi.org/10.1371/journal.pone.0186023

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