Transcriptional profiling analysis of Penicillium digitatum, the causal agent of citrus green mold, unravels an inhibited ergosterol biosynthesis pathway in response to citral

Abstract

Background: Green mold caused by Penicillium digitatum is the most damaging postharvest diseases of citrus fruit. Previously, we have observed that citral dose-dependently inhibited the mycelial growth of P. digitatum, with the minimum inhibitory concentration (MIC) of 1.78 mg/mL, but the underlying molecular mechanism is barely understood.

Results: In this study, the transcriptional profiling of the control and 1/2MIC-citral treated P. digitatum mycelia after 30 min of exposure were analyzed by RNA-Seq. A total of 6355 genes, including 2322 up-regulated and 4033 down-regulated genes, were found to be responsive to citral. These genes were mapped to 155 KEGG pathways, mainly concerning mRNA surveillance, RNA polymerase, RNA transport, aminoacyl-tRNA biosynthesis, ABC transporter, glycolysis/gluconeogenesis, citrate cycle, oxidative phosphorylation, sulfur metabolism, nitrogen metabolism, inositol phosphate metabolism, fatty acid biosynthesis, unsaturated fatty acids biosynthesis, fatty acid metabolism, and steroid biosynthesis. Particularly, citral exposure affected the expression levels of five ergosterol biosynthetic genes (e.g. ERG7, ERG11, ERG6, ERG3 and ERG5), which corresponds well with the GC-MS results, the reduction in ergosterol content, and accumulation of massive lanosterol. In addition, ERG11, the gene responsible for lanosterol 14α-demethylase, was observed to be the key down-regulated gene in response to citral.

Conclusion: Our present finding suggests that citral could exhibit its antifungal activity against P. digitatum by the down-regulation of ergosterol biosynthesis.

Keywords: Citral; Ergosterol biosynthesis; Penicillium digitatum; RNA-Seq.

Fig. 1

a The growth curve of P. digitatum mycelia; b The wet weight of P. digitatum mycelia exposed to citral, (○) CK; (●) 1/2MIC; (▲) MIC. Values are the mean ± SD of three measurements

Fig. 2

The length distribution of unigenes after de novo assembly using the reads from CK30, T30, and CK30&T30 samples, the horizontal coordinates are unigene lengths and the vertical coordinates are numbers of unigenes

Fig. 3

Species distribution of the BLASTx matches (with an E-value < 10⁻⁵) to the unique transcripts against the non-redundant protein database. a The E-value distribution of NR annotation result; b The similarity distribution of NR annotation result; c The species distribution of NR annotation result

Fig. 4

GO classification analysis of unigenes in all-unigene. GO functions is showed in X-axis. The right Y-axis shows the number of genes which has the GO function, and the left Y-axis shows the percentage

Fig. 5

Clusters of orthologous groups (COG) function classification of unigenes in all-unigene. The horizontal coordinates are COG function classes, and the vertical coordinates are numbers of unigenes in one class. The notation on the right is the full name of the functions in X-axis

Fig. 6

Factors and intermediates of fungal ergosterol biosynthetic pathway

Fig. 7

Changes in the expression of ergosterol biosynthesis genes of P. digitatum mycelia treated by CK and 1/2MIC citral for 0, 30, 60 and 120 min, (○) CK; (●) 1/2MIC. Values are the mean ± SD of three measurements

Fig. 8

HPLC analysis of ergosterol in control and 1/2MIC citral treated samples. a: The ergosterol standard (with a retention time of 9.376 min); b: The ergosterol in control samples (with a retention time of 9.205 min); c: The ergosterol in 1/2MIC citral treated samples (with a retention time of 9.269 min); d: Total ergosterol contents in control (○) and 1/2MIC citral treated (●) P. digitatum cells, and values are the mean ± SD of three measurements

Fig. 9

HPLC analysis of lanosterol in control and 1/2MIC citral treated samples. a: The lanosterol standard (with a retention time of 11.669 min); b: The lanosterol in control samples (with a retention time of 12.426 min); c: The lanosterol in 1/2MIC citral treated samples (with a retention time of 12.288 min); d: Total lanosterol contents in control (○) and 1/2MIC citral treated (●) P. digitatum cells, and values are the mean ± SD of three measurements