Transcriptome analysis of fungicideresponsive gene expression profiles in two Penicillium italicum strains with different response to the sterol demethylation inhibitor (DMI) fungicide prochloraz

- Functional enrichments identified 15 DEGs in the prochloraz-induced Pi-R transcriptome, simultaneously up-regulated in P.
- Intriguingly, some strategies adopted by the present Pi-R were not observed in the previously documented prochloraz-resistant P.
- However, resistance to these DMI fungicides has frequently occurred for the Penicillium molds in the past decade, especially for P.
- The site mutations in CYP51s (ERG11-encoding proteins) can alter drug-target interactions and increase DMI-resistance for various fungal pathogens, as reported in the model yeast Saccharomyces cerevisiae the clinical pathogens Candida albicans [17 – 20] and Aspergillus fumigatus and the plant pathogens Mycosphaerella graminicola [24, 25], Monilinia fructicola [26] and P.
- To date, the MATE contribution to fungal drug-resistance was only reported in the ectomycorrhizal fungus Tricholoma vaccinum [62] and the citrus patho- genic fungus P.
- The over- expression of CaMK2 (also named Cmk2) in the yeast S..
- italicum strains with and without prochloraz treatment, to identify differentially expressed genes (DEGs) involved in the azole-class fungicide resistance, and to provide theoret- ical cues to explain P.
- In the present study, Pi-R and Pi-S were treated with or without DMI-fungicide prochloraz to prepare four RNA- seq samples, i.e., Pi-R-I, Pi-R-NI, Pi-S-I and Pi-S-NI..
- All unigene expression levels in the four libraries were classified into five intervals, ac- cording to FPKM values (Table 1), and more than 50%.
- Based on the above FPKM values, hierarchical cluster (i.e., heat map) analysis was performed to visualize DEG profiles between Pi-R-I, Pi-R-NI, Pi-S-I and Pi-S-NI libraries (Fig.
- Noticeably, pro- chloraz induced more dramatic change in gene expres- sion profile between Pi-R-I and Pi-R-NI than between Pi-S-I and Pi-S-NI, suggesting the involvement of more DEGs in Pi-R response to prochloraz..
- 1 were set as cut-off standard to identify DEGs between different librar- ies, including a) Pi-R-I vs Pi-R-NI, b) Pi-S-I vs Pi-S-NI, c) Pi-R-I vs Pi-S-I, and d) Pi-R-NI vs Pi-S-NI (Fig.
- We identified 1) 1052 DEGs between Pi-R-I and Pi-R-NI (614 up-regulated and 438 down-regulated) (Fig.
- 2) 298 DEGs between Pi-S-I and Pi-S-NI (63 up-regulated and 235 down- regulated) (Fig.
- 3) 1482 DEGs between Pi-R-I and Pi-S-I (811 up- regulated and 671 down-regulated) (Fig.
- and 4) 958 DEGs between Pi-R-NI and Pi-S-NI (422 up-regulated and 536 down-regulated) (Fig..
- FPKM interval Pi-R-I Pi-R-NI Pi-S-I Pi-S-NI.
- Based on the volcano plot analysis, we applied Venn diagrams to profile the DEG distribution between Pi-R-I vs Pi-R-NI and Pi-S-I vs Pi-S- NI (Fig.
- 3a) and between Pi-R-I vs Pi-S-I and Pi-R-NI vs Pi-S-NI (Fig.
- 3b, the overlap part of circles Pi-R-I vs Pi-S-I and Pi-R-NI vs Pi-S-NI comprised 513 DEGs that might represent DEGs irrelevant to prochloraz induction.
- In contrast, only 110 DEGs were distributed in the overlap part of circles Pi-R-I vs Pi-R-NI and Pi-S-I vs Pi-S-NI (Fig.
- The number of total GO terms and its distribution in the three categories for each comparison are listed in Table 3.
- In the comparison Pi-R-I vs Pi-R-NI (Fig.
- In the comparison Pi-S-I vs Pi-S-NI (Fig.
- In the comparison Pi-R-I vs Pi-S-I (Fig.
- In the comparison Pi-R-NI vs Pi-S-NI (Fig.
- Figure 5 re- ports the distribution of up- and down-regulated unigenes in the top 30 enriched GO terms for the 4 comparisons mentioned above.
- Interestingly, the DEGs enriched in the top 30 GO terms were found mostly up-regulated in the comparisons Pi-R-I vs Pi-R-NI and Pi-R-I vs Pi-S-I (Figs..
- 5a and c) and generally down-regulated in the compari- sons Pi-S-I vs Pi-S-NI and Pi-R-NI vs Pi-S-NI (Figs.
- In the present four comparisons, KEGG analysis enriched prochloraz- responsive DEGs into only two pathways, i.e., steroid.
- 083360) in the comparisons Pi-R (I/NI) and Pi-S (I/NI), ERG2 (PITC_020620) in the comparisons Pi-R (I/NI) and Pi-S (I/NI), and ERG6 (PITC_014340) in the com- parisons Pi-R (I/NI) and I (Pi-R/Pi-S).
- 2 Volcano plot of DEGs in the comparison between Pi-R-I and Pi-R-NI (a), Pi-S-I and Pi-S-NI (b), Pi-R-I and Pi-S-I (c), and Pi-R-NI and Pi-S-NI (d)..
- Pi-R-I vs Pi-R-NI 16 6 37 0.
- Pi-S-I vs Pi-S-NI 8 1 19 0.
- Pi-R-I vs Pi-S-I 19 12 68 0.
- Pi-R-NI vs Pi-S-NI 9 11 57 0.
- Additionally, FPKM-based unigene ex- pression quantification combined with local Blast-based annotation revealed differential expression patterns for particular prochloraz-responsive unigenes in the present 4 comparisons, including CYP51B (PITC_064600), CYP51C (PITC_028940), Bck1 (PITC_061930) and Slt2 (PITC_008290) (Additional file 10: Table S8).
- Consider- ing 1) functional clustering of CYP51A/B/C (i.e., iso- froms of drug-target gene CYP51) and 2) cascaded association of Bck1 (encoding MAPKKK), Mkk1 (encod- ing MAPKK) and Slt2 (encoding MAPK) in Slt2-MAPK pathway, we also performed qRT-PCR validation for the 4 prochloraz-responsive unigenes that were not included in the present DEG list for comparison (i.e., not included in Additional files 5-8: Tables S3–6).
- 3 Venn diagram of DEGs shared in DEG groups Pi-R-I vs Pi-R-NI and Pi-S-I vs Pi-S-NI (a) and DEG groups Pi-R-I vs Pi-S-I and Pi-R-NI vs Pi-S-NI (b).
- Yellow circle stands for number of DEGs between Pi-R-I and Pi-S-I (a) and between Pi-R-I and Pi-R-NI (b).
- Purple circle represents number of DEGs between Pi-R-NI and Pi-S-NI (a) and between Pi-S-I and Pi-S-NI (b).
- The overlapping region comprises the DEGs shared in the two DEG groups Pi-R-I vs Pi-R-NI and Pi-S-I vs Pi- S-NI (a) and another two DEG groups Pi-R-I vs Pi-S-I and Pi-R-NI vs Pi-S-NI (b).
- Pi-R-I vs Pi-R-NI .
- Pi-S-I vs Pi-S-NI .
- Pi-R-I vs Pi-S-I .
- Pi-R-NI vs Pi-S-NI .
- The Table lists term numbers in GO enrichment and in the three GO categories, i.e., Biological Process (BP), Cellular Component (CC), and Molecular Function (MF), for each comparison in the present study, and correspondingly, also lists differentially expressed gene (DEG) numbers.
- In the past decades, conventional synthetic DMI- fungicides, such as prochloraz and imazalil, were widely applied to control Penicillium decay, but undesirably, a considerable number of resistant isolates including P..
- Spe- cially, ABC and MFS transporter-encoding genes, each containing multiple isoforms, were reported to be simul- taneously up-regulated in the prochloraz-resistant P..
- Each GO category (type) displays 30 terms (listed on Y-axis) significantly or most enriched for DEGs in the given comparisons, and X-axis indicates the number of DEGs involved in particular GO term.
- 4) was also observed in the prochloraz-treated Pi-R ra- ther than Pi-S (Table 4 and Fig.
- Interestingly, regarding these drug-efflux pump-encoding genes, over-expressed in the prochloraz- induced P.
- italicum transcriptome, their orthologous genes in the prochloraz-induced P.
- More interestingly, regarding another class of drug- pump-encoding genes, i.e., MATEs, none was found to be differentially expressed in the prochloraz-induced Pi-R and Pi-S transcriptomes (Table 2).
- The orthologous gene of PdMATE3 has been identified in the present Pi-R and Pi-S genomes, however, no mRNA transcripts of this gene was detected in the prochloraz-treated P.
- 5 Distribution of up- and down-regulated genes in the top 30 enriched GO terms for Pi-R-I vs Pi-R-NI (a), Pi-S-I vs Pi-S-NI (b), Pi-R-I vs Pi-S-I (c), and Pi-R-NI vs Pi-S-NI (d).
- GO ID (term) DEG ID DEG name (GO-annotated) Fold change (log2) of DEG expression in the.
- Pi-R (I/NI), Pi-S (I/NI), I (Pi-R/Pi-S) and NI (Pi-R/Pi-S) indicate comparisons Pi-R-I vs Pi-R-NI, Pi-S-I vs Pi-S-NI, Pi-R-I vs Pi-S- I and Pi-R-NI vs Pi-S-NI, respectively.
- indicates the unfeasibility to generate log2(fold change) when the DEG is not included in the comparison.
- But such transcriptomic response of MATE transporter-encoding genes did not occur in the present P.
- 6b), Table 5 Summary of KEGG enrichments for prochloraz-responsive DEGs in the present 4 comparisons.
- All values obtained in the qRT-PCR analysis were expressed as the mean ± SD from 5 biological repeats each containing 3 technical replicates, and independent samples t-test (n = 5) was applied in the SPSS Statistics 17.0 context to assess the significance of differences between the means (*p <.
- However, according to the prochloraz-induced P.
- digitatum transcriptome [11], the orthologous genes of CYP51B and CYP51C in the prochloraz-resistant P.
- ERG2 and ERG6, the other two ERGs in ergosterol-biosynthesis pathway, as recommended to be potential multidrug targets [89], were up-regulated in the prochloraz-treated Pi-R (Tables 4, 5 and Fig.
- Such ERGs expression profile was observed in the DMI-resistant P.
- italicum, but not in the prochloraz- resistant P.
- The Slt2-MAPK middle-stream components, two MAPKK-encoding genes ScMkk1 and ScMkk2, have been characterized in the model yeast S.
- In the present study, Mkk1 (PITC_088710), the homolog of ScMkk1 in P.
- italicum species, was GO- and KEGG-enriched as up-regulated DEG in the prochloraz- treated Pi-R (Tables 4 and 5), indicating the involvement of Slt2-MAPK in the P.
- The difference in CaMK response to DMI-fungicides in the two Penicil- lium species needs further research..
- Some differ- ences in the choice of anti-DMI strategy between P..
- In the present study, DEGs were selected as transcripts and/or unigenes differentially expressed with at least 2-fold change (i.e., the absolute value of log2 Fold change ≥1.0) and corrected P-value.
- Nineteen unigenes related to azole-drug resistance in the present P.
- Relative ratios for the expression of each selected unigene were further calculated in the 4 comparison groups, including Pi-R (I/NI) (i.e., Pi-R-I relative to Pi-R-NI), Pi-S (I/NI) (i.e., Pi-S-I relative to Pi-S-NI), I (Pi-R/Pi-S) (i.e., Pi-R-I relative to Pi-S-I), and NI (Pi-R/Pi-S) (i.e., Pi-R-NI rela- tive to Pi-S-NI).
- All values obtained in the qRT-PCR analysis were expressed as the mean ± SD (standard deviation of the mean), and based on 5 independent ex- periments (i.e., 5 biological repeats), independent sam- ples t-test (n = 5) was applied in the SPSS Statistics 17.0 context to assess the significance of differences between the means (*p <.
- Primers used in the present qRT-PCR validation..
- Summary of DEGs in the comparison between prochloraz-induced and no-induced Pi-R strains..
- Summary of DEGs in the comparison between prochloraz-induced and no-induced Pi-S strains..
- Summary of DEGs in the comparison between Pi-R and Pi-S strains under prochloraz-induced conditions..
- Summary of DEGs in the comparison between Pi-R and Pi-S strains without prochloraz induction..
- Summary of distribution (hit records) of 19 selected DEGs towards GO enrichments in the present 4 comparisons..
- FPKM values of the additional 4 prochloraz-responsive unigenes in the present comparisons..
- The funding body didn ’ t play any roles in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript..
- A novel ATP-binding cassette transporter involved in multidrug resistance in the phytopathogenic fungus Penicillium digitatum.
- Amino acid substitutions in the cytochrome P-450 lanosterol 14 α -demethylase (CYP51A1) from azole- resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents.
- Contribution of mutations in the cytochrome P450 14 α -demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans.
- A newly identified amino acid substitution T123I in the 14 α -demethylase (Erg11p) of Candida albicans confers azole resistance.
- The point mutation G461S in the MfCYP51 gene is associated with tebuconazole resistance in Monilinia fructicola populations in Brazil.
- Imazalil resistance linked to a unique insertion sequence in the PdCYP51 promoter region of Penicillium digitatum.
- QTL mapping of fungicide sensitivity reveals novel genes and pleiotropy with melanization in the pathogen Zymoseptoria tritici.
- A novel ABC transporter gene, PMR5, is involved in multidrug resistance in the phytopathogenic fungus Penicillium digitatum.
- PdMfs1, a major facilitator superfamily transporter from Penicillium digitatum, is partially involved in the imazalil-resistance and pathogenicity.
- Multidrug efflux transporters in the MATE family..
- A transporter for abiotic stress and plant metabolite resistance in the ectomycorrhizal fungus Tricholoma vaccinum.
- The glutathione peroxidase-mediated reactive oxygen species resistance, fungicide sensitivity and cell wall construction in the citrus fungal pathogen Alternaria alternata.
- A major facilitator superfamily transporter-mediated resistance to oxidative stress and fungicides requires Yap1, Skn7, and MAP kinases in the citrus fungal pathogen Alternaria alternata.
- The stress-activated protein kinase FgOS- 2 is a key regulator in the life cycle of the cereal pathogen Fusarium graminearum.
- calmodulin-dependent protein kinases are involved in the regulation of cell wall integrity and oxidative stress response in Candida albicans.
- MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C

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