MYB24, MYB144, and MYB168 positively regulate suberin biosynthesis at potato tuber wounds during healing.

The essence of wound healing is the accumulation of suberin at wounds, which is formed by suberin polyphenolic (SPP) and suberin polyaliphatic (SPA). The biosynthesis of SPP and SPA monomers is catalyzed by several enzyme classes related to phenylpropanoid metabolism and fatty acid metabolism, respectively. However, how suberin biosynthesis is regulated at the transcriptional level during potato (Solanum tuberosum) tuber wound healing remains largely unknown. Here, 6 target genes and 15 transcription factors related to suberin biosynthesis in tuber wound healing were identified by RNA-seq technology and qRT-PCR. Dual luciferase and yeast one-hybrid assays showed that StMYB168 activated the target genes StPAL, StOMT, and St4CL in phenylpropanoid metabolism. Meanwhile, StMYB24 and StMYB144 activated the target genes StLTP, StLACS, and StCYP in fatty acid metabolism, and StFHT involved in the assembly of SPP and SPA domains in both native and wound periderms. More importantly, virus-induced gene silencing in S. tuberosum and transient overexpression in Nicotiana benthamiana assays confirmed that StMYB168 regulates the biosynthesis of free phenolic acids, such as ferulic acid. Furthermore, StMYB24/144 regulated the accumulation of suberin monomers, such as ferulates, α, ω-diacids, and ω-hydroxy acids. In conclusion, StMYB24, StMYB144, and StMYB168 have an elaborate division of labor in regulating the synthesis of suberin during tuber wound healing.

Bacterial-fungal crosstalk is defined by a fungal lactone mycotoxin and its degradation by a bacterial lactonase.

Bacteria, fungi, and mammals contain lactonases that can degrade the Gram-negative bacterial quorum sensing (QS) molecules N-acyl homoserine lactones (AHLs). AHLs are critical for bacteria to coordinate gene expression and pathogenicity with population density. However, AHL-degrading lactonases present variable substrate ranges, including degradation of the Pencillium expansum lactone mycotoxin patulin. We selected Erwinia spp. as our model bacteria to further investigate this interaction. We find both native apple microbiome Erwinia spp. and the fruit tree pathogen Erwinia amylovora to be inhibited by patulin. At patulin concentrations that inhibited E. amylovora growth, expression of E. amylovora lactonase encoded by EaaiiA was increased. EaAiiA demonstrated the ability to degrade patulin in vitro, as well, as in vivo where it reduced apple disease and patulin production by P. expansum. Fungal-bacterial co-cultures revealed that the E. amylovora Δeaaiia strain failed to protect apples from P. expansum infections, which contained significant amounts of patulin. Our results suggest that bacterial lactonase production can modulate the pathogenicity of P. expansum in response to the secretion of toxic patulin. IMPORTANCE: Chemical signaling in the microbial world facilitates the regulation of gene expression as a function of cell population density. This is especially true for the Gram-negative bacterial signal N-acyl homoserine lactone (AHL). Lactonases that deactivate AHLs have attracted a lot of attention because of their antibacterial potential. However, the involvement of these enzymes in inhibiting fungal pathogens and the potential role of these enzymes in bacterial-fungal interactions are unknown. Here, we find that a bacterial enzyme involved in the degradation of AHLs is also induced by and degrades the fungal lactone mycotoxin, patulin. This work supports the potential use of bacterial enzymes and/or the producing bacteria in controlling the post-harvest fruit disease caused by the patulin-producing fungus Penicillium expansum.

The small GTPase Ypt7 of Penicillium expansum is required for growth, patulin biosynthesis and virulence.

Ypt GTPases are the largest subfamily of small GTPases involved in membrane transport. Here, a PeYpt7 gene deletion mutant of P. expansum was constructed. The ΔPeYpt7 mutant showed reduced colony growth with abnormal mycelial growth, reduced conidiation, and insufficient spore development. The mutation rendered the pathogen susceptible to osmotic stress and cell wall stressors. In addition, the absence of PeYpt7 reduced patulin production in P. expansum and significantly limited gene expression (PatG, PatH, PatI, PatD, PatF, and PatL). In addition, the mutant showed attenuated virulence in infected fruit and reduced expression of pathogenic factors was (PMG, PG, PL, and GH1). Thus, PeYpt7 modulates the growth, morphology, patulin accumulation, and pathogenicity of P. expansum by limiting the expression of related genes.

ROS mediated by TrPLD3 of Trichothecium roseum participated cell membrane integrity of apple fruit by influencing phosphatidic acid metabolism.

Trichothecium roseum is a typical necrotrophic fungal pathogen that not only bring about postharvest disease, but contribute to trichothecenes contamination in fruit and vegetables. Phospholipase D (PLD), as an important membrane lipid degrading enzyme, can produce phosphatidic acid (PA) by hydrolyzing phosphatidylcholine (PC) and phosphatidylinositol (PI). PA can promote the production of reactive oxygen species (ROS) by activating the activity of NADPH oxidase (NOX), thereby increasing the pathogenicity to fruit. However, the ROS mediated by TrPLD3 how to influence T. roseum infection to fruit by modulating phosphatidic acid metabolism, which has not been reported. In this study, the knockout mutant and complement strain of TrPLD3 were constructed through homologous recombination, TrPLD3 was tested for its effect on the colony growth and pathogenicity of T. roseum. The experimental results showed that the knockout of TrPLD3 inhibited the colony growth of T. roseum, altered the mycelial morphology, completely inhibited the sporulation, and reduced the accumulation of T-2 toxin. Moreover, the knockout of TrPLD3 significantly decreased pathogenicity of T. roseum on apple fruit. Compared to inoculated apple fruit with the wide type (WT), the production of ROS in apple infected with ΔTrPLD3 was slowed down, the relative expression and enzymatic activity of NOX, and PA content decreased, and the enzymatic activity and gene expression of superoxide dismutase (SOD) increased. In addition, PLD, lipoxygenase (LOX) and lipase activities were considerably decreased in apple fruit infected with ΔTrPLD3, the changes of membrane lipid components were slowed down, the decrease of unsaturated fatty acid content was alleviated, and the accumulation of saturated fatty acid content was reduced, thereby maintaining the cell membrane integrity of the inoculated apple fruit. We speculated that the decreased PA accumulation in ΔTrPLD3-inoculated apple fruit further weakened the interaction between PA and NOX on fruit, resulting in the reduction of ROS accumulation of fruits, which decreased the damage to the cell membrane and maintained the cell membrane integrity, thus reducing the pathogenicity to apple. Therefore, TrPLD3-mediated ROS plays a critical regulatory role in reducing the pathogenicity of T. roseum on apple fruit by influencing phosphatidic acid metabolism.

The histone demethylase KdmB is part of a trimeric protein complex and mediates virulence and mycotoxin production in Penicillium expansum.

Epigenetic modification of chromosome structure has increasingly been associated with alterations in secondary metabolism and sporulation defects in filamentous fungal pathogens. Recently, the epigenetic reader protein SntB was shown to govern virulence, spore production and mycotoxin synthesis in the fruit pathogen Penicillium expansum. Through immunoprecipitation-coupled mass spectrometry, we found that SntB is a member of a protein complex with KdmB, a histone demethylase and the essential protein RpdA, a histone deacetylase. Deletion of kdmB phenocopied some but not all characteristics of the ΔsntB mutant. KdmB deletion strains exhibited reduced lesion development on Golden Delicious apples and this was accompanied by decreased production of patulin and citrinin in host tissue. In addition, ΔkdmB mutants were sensitive to several cell wall stressors which possibly contributed to the decreased virulence observed on apples. Slight differences in spore production and germination rates of ΔkdmB mutants in vitro did not impact overall diameter growth in culture.

AaCaMKs Positively Regulate Development, Infection Structure Differentiation and Pathogenicity in Alternaria alternata, Causal Agent of Pear Black Spot.

Calcium/calmodulin-dependent protein kinase (CaMK), a key downstream target protein in the Ca2+ signaling pathway of eukaryotes, plays an important regulatory role in the growth, development and pathogenicity of plant fungi. Three AaCaMKs (AaCaMK1, AaCaMK2 and AaCaMK3) with conserved PKC_like superfamily domains, ATP binding sites and ACT sites have been cloned from Alternaria alternata, However, their regulatory mechanism in A. alternata remains unclear. In this study, the function of the AaCaMKs in the development, infection structure differentiation and pathogenicity of A. alternata was elucidated through targeted gene disruption. The single disruption of AaCaMKs had no impact on the vegetative growth and spore morphology but significantly influenced hyphae growth, sporulation, biomass accumulation and melanin biosynthesis. Further expression analysis revealed that the AaCaMKs were up-regulated during the infection structure differentiation of A. alternata on hydrophobic and pear wax substrates. In vitro and in vivo analysis further revealed that the deletion of a single AaCaMKs gene significantly reduced the A. alternata conidial germination, appressorium formation and infection hyphae formation. In addition, pharmacological analysis confirmed that the CaMK specific inhibitor, KN93, inhibited conidial germination and appressorium formation in A. alternata. Meanwhile, the AaCaMKs genes deficiency significantly reduced the A. alternata pathogenicity. These results demonstrate that AaCaMKs regulate the development, infection structure differentiation and pathogenicity of A. alternata and provide potential targets for new effective fungicides.

Transcriptome sequencing and differential expression analysis of natural and BTH-treated wound healing in potato tubers (Solanum tuberosum L.).

BACKGROUND: Wound healing is a representative phenomenon of potato tubers subjected to mechanical injuries. Our previous results found that benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) promoted the wound healing of potato tubers. However, the molecular mechanism related to inducible wound healing remains unknown. RESULTS: Transcriptomic evaluation of healing tissues from potato tubers at three stages, namely, 0 d (nonhealing), 5 d (wounded tubers healed for 5 d) and 5 d (BTH-treated tubers healed for 5 d) using RNA-Seq and differentially expressed genes (DEGs) analysis showed that more than 515 million high-quality reads were generated and a total of 7665 DEGs were enriched, and 16 of these DEGs were selected by qRT-PCR analysis to further confirm the RNA sequencing data. Gene ontology (GO) enrichment analysis indicated that the most highly DEGs were involved in metabolic and cellular processes, and KEGG enrichment analysis indicated that a large number of DEGs were associated with plant hormones, starch and sugar metabolism, fatty acid metabolism, phenylpropanoid biosynthesis and terpenoid skeleton biosynthesis. Furthermore, a few candidate transcription factors, including MYB, NAC and WRKY, and genes related to Ca2+-mediated signal transduction were also found to be differentially expressed during wound healing. Most of these enriched DEGs were upregulated after BTH treatment. CONCLUSION: This comparative expression profile provided useful resources for studies of the molecular mechanism via these promising candidates involved in natural or elicitor-induced wound healing in potato tubers.

The pH signalling transcription factor PacC modulate growth, development, stress response and pathogenicity of Trichothecium roseum.

pH is one of the important environmental factors that affect the growth, development and pathogenicity of postharvest pathogen. The transcription factor PacC dominates the pH signal pathway. PacC in Trichothecium roseum showed three typical conserved zinc finger domains and closest homology to Fusarium graminearum. T. roseum increased the environmental pH both in vitro and in vivo. Expression patterns of TrpacC under different pH showed that at increasing pH from 3 to 5, the wild-type (WT) strain induced the expression of TrPacC in parallel to increased fungal growth; however, TrPacC expression decline at higer pH than 5, while fungal growth continued to increase. Development of a ΔTrPacC mutant down-regulated the expression of TrbrlA, TrabaA and TrwetA, reduced sporulation and delayed spore germination, resulting in smaller spores and sparse hyphae. ΔTrPacC mutant was sensitive to ionic stress, oxidative stress and cell wall integrity stress compared to the WT strain, especially the ionic stress. In addition, ∆TrPacC mutant showed reduced pathogenicity to muskmelon and tomato fruits. Taken together, T. roseum is an alkalinizing fungus, and the acidic environment could induce TrPacC expression. TrPacC positively regulates fungal growth and development as well as pathogenicity showing effect on fungal response to different stresses.

BcHnm1, a predicted choline transporter, modulates conidial germination and virulence in Botrytis cinerea.

Previous network-based comparative genomic analysis between major lifestyles of fungal plant pathogens highlighted that HNM1, a predicted choline transporter, is part of the necrotroph core-genome's functions. In this work we have generated and characterized deletion mutants and developed complemented strains for the HNM1 homolog (Bchnm1) in the necrotrophic model fungal plant pathogen Botrytis cinerea. The Bchnm1 deletion mutants exhibited reduced conidia germination and germ tube elongation. The functional activity of the Δbchnm1 deletion mutants was illustrated by reduced necrotic colonization of B. cinerea on tomato and French bean leaves. The role of BcHnm1 in germination was also supported by qRT-PCR results that illustrated increased Bchnm1 transcript levels during the early infection stages (at 16 h post inoculation) of the WT strain on tomato plant leaves, and during conidia germination (in-vitro). In line with the predicted function of BcHnm1 in choline transport, Δbchnm1 deletion mutant showed an attenuated choline import capacity. The potential role of choline in the WT B. cinerea was further demonstrated by an increase in conidia germination (by 100%) in the presence of 1 mM exogenous choline while growth in the presence of hemicholinium-3, an inhibitor of choline transporter, showed 40% inhibition in germination. In contrast to the WT, exogenous choline and the inhibitor did not affect conidia germination in the Δbchnm1 deletion mutants. Collectively, this study shows for the first time that BcHnm1, a predicted choline transporter, is important for conidial germination, germ tube elongation, response to exogenous choline, and virulence in plant pathogenic fungi.

AaCaM is required for infection structure differentiation and secondary metabolites in pear fungal pathogen Alternaria alternata.

AIMS: Calmodulin (CaM), acts as a kind of multifunctional Ca2+ sensing protein, which is ubiquitous in fungi, is highly conserved across eukaryotes and is involved in the regulation of a range of physiological processes, including morphogenesis, reproduction and secondary metabolites biosynthesis. Our aim was to understand the characteristics and functions of AaCaM in Alternaria alternata, the causal agent of pear black spot. METHODS AND RESULTS: A 450 bp cDNA sequence of AaCaM gene of A. alternata was cloned by the PCR homology method. Sequence analysis showed that this protein encoded by AaCaM was a stable hydrophilic protein and had a high similarity to Neurospora crassa (CAA50271.1) and other fungi. RT-qPCR analysis determined that AaCaM was differentially upregulated during infection structural differentiation of A. alternata both on hydrophobic and pear wax extract-coated surface, with a 3.37-fold upregulation during the hydrophobic induced appressorium formation period (6 h) and a 1.46-fold upregulation during the infection hyphae formation period (8 h) following pear wax induction. Pharmaceutical analysis showed that the CaM-specific inhibitor, trifluoperazine (TFP), inhibited spore germination and appressorium formation, and affected toxins and melanin biosynthesis in A. alternata. CONCLUSIONS: AaCaM plays an important role in regulating infection structure differentiation and secondary metabolism of A. alternata. SIGNIFICANCE AND IMPACT OF STUDY: Our study provides a theoretical basis for further in-depth investigation of the specific role of AaCaM in the calcium signalling pathway underlying hydrophobic and pear wax-induced infection structure differentiation and pathogenicity of A. alternata.

Molecular Characterization of Phospholipase C in Infection Structure Differentiation Induced by Pear Fruit Surface Signals, Stress Responses, Secondary Metabolism, and Virulence of Alternaria alternata.

Fungal pathogens use plant surface physiochemical signals to trigger specific developmental processes. To assess the role of phospholipase C (PLC) in mediating plant stimuli sensing of Alternaria alternata, the function of three PLC genes was characterized by constructing ΔAaPLC mutants. Here we showed that fruit wax-coated surfaces significantly induced appressorium formation in A. alternata and mutants. Germination of ΔAaPLC mutants did not differ from the wild type. Deletion of AaPLC1 led to the decrease of appressorium formation and infected hyphae, but the degree of reduction varies between the different types of waxes, with the strongest response to pear wax. Appressorium formation and infected hyphae of the ΔAaPLC1 mutant on dewaxed onion epidermis mounted with pear wax (θ4) were reduced by 14.5 and 65.7% after 8 h incubation, while ΔAaPLC2 and ΔAaPLC3 formed the same infection hyphae as wild type. In addition, AaPLC1 mutation caused pleiotropic effects on fungal biological function, including growth deficiency, changes in stress tolerance, weakening of pathogenicity to the host, as well as destruction of mycotoxin synthesis. Both AaPLC2 and AaPLC3 genes were found to have some effects on stress response and mycotoxin production. Taken together, AaPLC genes differentially regulate the growth, stress response, pathogenicity, and secondary metabolism of A. alternata.

Transcriptome Profiling Data of Botrytis cinerea Infection on Whole Plant Solanum lycopersicum.

Botrytis cinerea is a foliar necrotrophic fungal-pathogen capable of infecting >580 genera of plants, is often used as model organism for studying fungal-host interactions. We used RNAseq to study transcriptome of B. cinerea infection on a major (worldwide) vegetable crop, tomato (Solanum lycopersicum). Most previous works explored only few infection stages, using RNA extracted from entire leaf-organ diluting the expression of studied infected region. Many studied B. cinerea infection, on detached organs assuming that similar defense/physiological reactions occurs in the intact plant. We analyzed transcriptome of the pathogen and host in 5 infection stages of whole-plant leaves at the infection site. We supply high quality, pathogen-enriched gene count that facilitates future research of the molecular processes regulating the infection process.

Sugar-regulated susceptibility of tomato fruit to Colletotrichum and Penicillium requires differential mechanisms of pathogenicity and fruit responses.

Colletotrichum gloeosporioides and Penicillium expansum cause postharvest diseases in tropical and deciduous fruit. During colonization, C. gloeosporioides and P. expansum secrete ammonia in hosts with low sugar content (LowSC) and gluconic acid in hosts with high sugar content (HighSC), respectively, as a mechanism to modulate enhanced pathogenicity. We studied the pathogens interactions with tomato lines of similar genetic background but differing in their sugar content. Colletotrichum gloeosporioides showed enhanced colonization of the LowSC line with differential expression response of 15% of its genes including enhanced relative expression of glycosyl hydrolases, glucanase and MFS-transporter genes. Enhanced colonization of P. expansum occurred in the HighSC line, accompanied by an increase in carbohydrate metabolic processes mainly phosphoenolpyruvate carboxykinase, and only 4% of differentially expressed genes. Gene response of the two host lines strongly differed depending on the sugar level. Limited colonization of HighSC line by C. gloeosporioides was accompanied by a marked alteration of gene expression compared the LowSC response to the same pathogen; while colonization by P. expansum resulted in a similar response of the two different hosts. We suggest that this differential pattern of fungal/host responses may be the basis for the differential of host range of both pathogens in nature.

Network analysis exposes core functions in major lifestyles of fungal and oomycete plant pathogens.

BACKGROUND: Genomic studies demonstrate that components of virulence mechanisms in filamentous eukaryotic pathogens (FEPs, fungi and oomycetes) of plants are often highly conserved, or found in gene families that include secreted hydrolytic enzymes (e.g., cellulases and proteases) and secondary metabolites (e.g., toxins), central to the pathogenicity process. However, very few large-scale genomic comparisons have utilized complete proteomes from dozens of FEPs to reveal lifestyle-associated virulence mechanisms. Providing a powerful means for exploration, and the discovery of trends in large-scale datasets, network analysis has been used to identify core functions of the primordial cyanobacteria, and ancient evolutionary signatures in oxidoreductases. RESULTS: We used a sequence-similarity network to study components of virulence mechanisms of major pathogenic lifestyles (necrotroph (ic), N; biotroph (ic), B; hemibiotroph (ic), H) in complete pan-proteomes of 65 FEPs and 17 saprobes. Our comparative analysis highlights approximately 190 core functions found in 70% of the genomes of these pathogenic lifestyles. Core functions were found mainly in: transport (in H, N, B cores); carbohydrate metabolism, secondary metabolite synthesis, and protease (H and N cores); nucleic acid metabolism and signal transduction (B core); and amino acid metabolism (H core). Taken together, the necrotrophic core contains functions such as cell wall-associated degrading enzymes, toxin metabolism, and transport, which are likely to support their lifestyle of killing prior to feeding. The biotrophic stealth growth on living tissues is potentially controlled by a core of regulatory functions, such as: small G-protein family of GTPases, RNA modification, and cryptochrome-based light sensing. Regulatory mechanisms found in the hemibiotrophic core contain light- and CO2-sensing functions that could mediate important roles of this group, such as transition between lifestyles. CONCLUSIONS: The selected set of enriched core functions identified in our work can facilitate future studies aimed at controlling FEPs. One interesting example would be to facilitate the identification of the pathogenic potential of samples analyzed by metagenomics. Finally, our analysis offers potential evolutionary scenarios, suggesting that an early-branching saprobe (identified in previous studies) has probably evolved a necrotrophic lifestyle as illustrated by the highest number of shared gene families between saprobes and necrotrophs.

Differential gene expression in tomato fruit and Colletotrichum gloeosporioides during colonization of the RNAi-SlPH tomato line with reduced fruit acidity and higher pH.

BACKGROUND: The destructive phytopathogen Colletotrichum gloeosporioides causes anthracnose disease in fruit. During host colonization, it secretes ammonia, which modulates environmental pH and regulates gene expression, contributing to pathogenicity. However, the effect of host pH environment on pathogen colonization has never been evaluated. Development of an isogenic tomato line with reduced expression of the gene for acidity, SlPH (Solyc10g074790.1.1), enabled this analysis. Total RNA from C. gloeosporioides colonizing wild-type (WT) and RNAi-SlPH tomato lines was sequenced and gene-expression patterns were compared. RESULTS: C. gloeosporioides inoculation of the RNAi-SlPH line with pH 5.96 compared to the WT line with pH 4.2 showed 30% higher colonization and reduced ammonia accumulation. Large-scale comparative transcriptome analysis of the colonized RNAi-SlPH and WT lines revealed their different mechanisms of colonization-pattern activation: whereas the WT tomato upregulated 13-LOX (lipoxygenase), jasmonic acid and glutamate biosynthesis pathways, it downregulated processes related to chlorogenic acid biosynthesis II, phenylpropanoid biosynthesis and hydroxycinnamic acid tyramine amide biosynthesis; the RNAi-SlPH line upregulated UDP-D-galacturonate biosynthesis I and free phenylpropanoid acid biosynthesis, but mainly downregulated pathways related to sugar metabolism, such as the glyoxylate cycle and L-arabinose degradation II. Comparison of C. gloeosporioides gene expression during colonization of the WT and RNAi-SlPH lines showed that the fungus upregulates ammonia and nitrogen transport and the gamma-aminobutyric acid metabolic process during colonization of the WT, while on the RNAi-SlPH tomato, it mainly upregulates the nitrate metabolic process. CONCLUSIONS: Modulation of tomato acidity and pH had significant phenotypic effects on C. gloeosporioides development. The fungus showed increased colonization on the neutral RNAi-SlPH fruit, and limited colonization on the WT acidic fruit. The change in environmental pH resulted in different defense responses for the two tomato lines. Interestingly, the WT line showed upregulation of jasmonate pathways and glutamate accumulation, supporting the reduced symptom development and increased ammonia accumulation, as the fungus might utilize glutamate to accumulate ammonia and increase environmental pH for better expression of pathogenicity factors. This was not found in the RNAi-SlPH line which downregulated sugar metabolism and upregulated the phenylpropanoid pathway, leading to host susceptibility.

Mutation of AREA affects growth, sporulation, nitrogen regulation, and pathogenicity in Colletotrichum gloeosporioides.

The GATA transcription factor AreA is a global nitrogen regulator that restricts the utilization of complex and poor nitrogen sources in the presence of good nitrogen sources in microorganisms. In this study, we report the biological function of an AreA homolog (the CgareA gene) in the fruit postharvest pathogen Colletotrichum gloeosporioides. Targeted gene deletion mutants of areA exhibited significant reductions in vegetative growth, increases in conidia production, and slight decreases in conidial germination rates. Quantitative RT-PCR (qRT-PCR) analysis revealed that the expression of AreA was highly induced under nitrogen-limiting conditions. Moreover, compared to wild-type and complemented strains, nitrogen metabolism-related genes were misregulated in ΔareA mutant strains. Pathogenicity assays indicated that the virulence of ΔareA mutant strains were affected by the nitrogen content, but not the carbon content, of fruit hosts. Taken together, our results indicate that CgareA plays a critical role in fungal development, conidia production, regulation of nitrogen metabolism and virulence in Colletotrichum gloeosporioides.

Fungal and host transcriptome analysis of pH-regulated genes during colonization of apple fruits by Penicillium expansum.

BACKGROUND: Penicillium expansum is a destructive phytopathogen that causes decay in deciduous fruits during postharvest handling and storage. During colonization the fungus secretes D-gluconic acid (GLA), which modulates environmental pH and regulates mycotoxin accumulation in colonized tissue. Till now no transcriptomic analysis has addressed the specific contribution of the pathogen's pH regulation to the P. expansum colonization process. For this purpose total RNA from the leading edge of P. expansum-colonized apple tissue of cv. 'Golden Delicious' and from fungal cultures grown under pH 4 or 7 were sequenced and their gene expression patterns were compared. RESULTS: We present a large-scale analysis of the transcriptome data of P. expansum and apple response to fungal colonization. The fungal analysis revealed nine different clusters of gene expression patterns that were divided among three major groups in which the colonized tissue showed, respectively: (i) differing transcript expression patterns between mycelial growth at pH 4 and pH 7; (ii) similar transcript expression patterns of mycelial growth at pH 4; and (iii) similar transcript expression patterns of mycelial growth at pH 7. Each group was functionally characterized in order to decipher genes that are important for pH regulation and also for colonization of apple fruits by Penicillium. Furthermore, comparison of gene expression of healthy apple tissue with that of colonized tissue showed that differentially expressed genes revealed up-regulation of the jasmonic acid and mevalonate pathways, and also down-regulation of the glycogen and starch biosynthesis pathways. CONCLUSIONS: Overall, we identified important genes and functionalities of P. expansum that were controlled by the environmental pH. Differential expression patterns of genes belonging to the same gene family suggest that genes were selectively activated according to their optimal environmental conditions (pH, in vitro or in vivo) to enable the fungus to cope with varying conditions and to make optimal use of available enzymes. Comparison between the activation of the colonized host's gene responses by alkalizing Colletotrichum gloeosporioides and acidifying P. expansum pathogens indicated similar gene response patterns, but stronger responses to P. expansum, suggesting the importance of acidification by P. expansum as a factor in its increased aggressiveness.

A Role of AREB in the Regulation of PACC-Dependent Acid-Expressed-Genes and Pathogenicity of Colletotrichum gloeosporioides.

Gene expression regulation by pH in filamentous fungi and yeasts is controlled by the PACC/RIM101 transcription factor. In Colletotrichum gloeosporioides, PACC is known to act as positive regulator of alkaline-expressed genes, and this regulation was shown to contribute to fungal pathogenicity. PACC is also a negative regulator of acid-expressed genes, however; the mechanism of downregulation of acid-expressed genes by PACC and their contribution to C. gloeosporioides pathogenicity is not well understood. RNA sequencing data analysis was employed to demonstrate that PACC transcription factor binding sites (TFBS) are significantly overrepresented in the promoter of PACC-upregulated, alkaline-expressed genes. In contrast, they are not overrepresented in the PACC-downregulated, acid-expressed genes. Instead, acid-expressed genes showed overrepresentation of AREB GATA TFBS in C. gloeosporioides and in homologs of five other ascomycetes genomes. The areB promoter contains PACC TFBS; its transcript was upregulated at pH 7 and repressed in ΔpacC. Furthermore, acid-expressed genes were found to be constitutively upregulated in ΔareB during alkalizing conditions. The areB mutants showed significantly reduced ammonia secretion and pathogenicity on tomato fruit. Present results indicate that PACC activates areB expression, thereby conditionally repressing acid-expressed genes and contributing critically to C. gloeosporioides pathogenicity.

Simultaneous transcriptome analysis of Colletotrichum gloeosporioides and tomato fruit pathosystem reveals novel fungal pathogenicity and fruit defense strategies.

The fungus Colletotrichum gloeosporioides breaches the fruit cuticle but remains quiescent until fruit ripening signals a switch to necrotrophy, culminating in devastating anthracnose disease. There is a need to understand the distinct fungal arms strategy and the simultaneous fruit response. Transcriptome analysis of fungal-fruit interactions was carried out concurrently in the appressoria, quiescent and necrotrophic stages. Conidia germinating on unripe fruit cuticle showed stage-specific transcription that was accompanied by massive fruit defense responses. The subsequent quiescent stage showed the development of dendritic-like structures and swollen hyphae within the fruit epidermis. The quiescent fungal transcriptome was characterized by activation of chromatin remodeling genes and unsuspected environmental alkalization. Fruit response was portrayed by continued highly integrated massive up-regulation of defense genes. During cuticle infection of green or ripe fruit, fungi recapitulate the same developmental stages but with differing quiescent time spans. The necrotrophic stage showed a dramatic shift in fungal metabolism and up-regulation of pathogenicity factors. Fruit response to necrotrophy showed activation of the salicylic acid pathway, climaxing in cell death. Transcriptome analysis of C. gloeosporioides infection of fruit reveals its distinct stage-specific lifestyle and the concurrent changing fruit response, deepening our perception of the unfolding fungal-fruit arms and defenses race.

Accumulation of the mycotoxin patulin in the presence of gluconic acid contributes to pathogenicity of Penicillium expansum.

Penicillium expansum, the causal agent of blue mold rot, causes severe postharvest fruit maceration through secretion of D-gluconic acid (GLA) and secondary metabolites such as the mycotoxin patulin in colonized tissue. GLA involvement in pathogenicity has been suggested but the mechanism of patulin accumulation and its contribution to P. expansum pathogenicity remain unclear. The roles of GLA and patulin accumulation in P. expansum pathogenicity were studied using i) glucose oxidase GOX2-RNAi mutants exhibiting decreased GOX2 expression, GLA accumulation, and reduced pathogenicity; ii) IDH-RNAi mutants exhibiting downregulation of IDH (the last gene in patulin biosynthesis), reduced patulin accumulation, and no effect on GLA level; and iii) PACC-RNAi mutants exhibiting downregulation of both GOX2 and IDH that reduced GLA and patulin production. Present results indicate that conditions enhancing the decrease in GLA accumulation by GOX2-RNAi and PACC-RNAi mutants, and not low pH, affected patulin accumulation, suggesting GLA production as the driving force for further patulin accumulation. Thus, it is suggested that GLA accumulation may modulate patulin synthesis as a direct precursor under dynamic pH conditions modulating the activation of the transcription factor PACC and the consequent pathogenicity factors, which contribute to host-tissue colonization by P. expansum.