Identification and functional analysis of Penicillium digitatum genes putatively involved in virulence towards citrus fruit

The fungus Penicillium digitatum, the causal agent of green mould rot, is the most destructive post-harvest pathogen of citrus fruit in Mediterranean regions. In order to identify P.digitatum genes up-regulated during the infection of oranges that may constitute putative virulence factors, we followed a polymerase chain reaction (PCR)-based suppression subtractive hybridization and cDNA macroarray hybridization approach. The origin of expressed sequence tags (ESTs) was determined by comparison against the available genome sequences of both organisms. Genes coding for fungal proteases and plant cell wall-degrading enzymes represent the largest categories in the subtracted cDNA library. Northern blot analysis of a selection of P.digitatum genes, including those coding for proteases, cell wall-related enzymes, redox homoeostasis and detoxification processes, confirmed their up-regulation at varying time points during the infection process. Agrobacterium tumefaciens-mediated transformation was used to generate knockout mutants for two genes encoding a pectin lyase (Pnl1) and a naphthalene dioxygenase (Ndo1). Two independent P.digitatum Δndo1 mutants were as virulent as the wild-type. However, the two Δpnl1 mutants analysed were less virulent than the parental strain or an ectopic transformant. Together, these results provide a significant advance in our understanding of the putative determinants of the virulence mechanisms of P.digitatum. Molecular Plant PathologyLGC research is funded in part by the Spanish Ministry of Economy and Innovation (AGL-2008-04828-C03-02 and AGL2011-30519-C03-01) and the Generalitat Valenciana (PROMETEO/2010/010, PROMETEOII/2014/027 and ACOMP/2011/250). MLP was supported by a FPI scholarship (BES-2006-12983). ARB acknowledges the support of the JAE-Doc program from CSIC cofounded by FSE.Peer Reviewe

The loss of the inducible Aspergillus carbonarius MFS transporter MfsA leads to ochratoxin A overproduction

Ochratoxin A (OTA), a nephrotoxic compound produced by certain Aspergillus and Penicillium species, is one of the most abundant mycotoxins in food commodities. Aspergillus carbonarius is the main source of OTA in wine, grape juice and dried vine fruits. Although many studies have focused on OTA production by A. carbonarius, little is known about the genes related to OTA production and transport. We have found a transporter that belongs to the major facilitator superfamily (MfsA) which is highly expressed with a 102-fold induction in an ochratoxigenic A. carbonarius strain compared to a low OTA producer strain. The encoding mfsA gene shows similarity to the multidrug efflux transporter flu1 from Candida albicans. A high number of putative transcription factor binding sites involved in the response to stress were identified within the promoter of mfsA. Phenotypical analysis of δ. mfsA deletion mutants revealed that the loss of mfsA leads to a slight growth reduction and increased OTA production. We therefore hypothesize that MfsA could be a stress response transporter whose disruption could cause an increase in oxidative stress together with a stimulation of mycotoxin production. •A. carbonarius mfsA expression is induced over 100 fold in an OTA producing strain.•mfsA gene has been cloned and sequenced.•An A. carbonarius δmfsA null mutant has been obtained by ATMT.•The δmfsA null mutants produce more OTA than the parental strain.•The expression of mfsA is up regulated by hydrogen peroxide.A. Crespo-Sempere was recipient of a FPI fellowship from the Spanish Government. This research was supported by grants AGL2005-00707 and AGL2008-04828-C03-02 from the Spanish Government.Peer Reviewe

The pH signaling transcription factor PacC is required for full virulence in Penicillium digitatum

Penicillium digitatum is the most important postharvest pathogen of citrus fruits. Along disease progression, the infected citrus peel tissue is acidified due to the accumulation of organic acids. So far, relatively little is known about the environmental factors that regulate pathogenicity in this fungus. In this study, the role of the pH signaling transcription factor PacC in the pathogenesis of P. digitatum was investigated. We identified the pacC ortholog (PdpacC) in P. digitatum and found that its transcript levels were elevated under alkaline conditions (pH ≥ 7) in vitro, as well as during the infection of citrus fruits in spite of the low pH (about 3.0 to 3.5) of the macerated tissue. Na+ and pectin also induced the expression of PdpacC. Disruption of PdpacC resulted in impaired mycelial growth under neutral or alkaline pH conditions and on synthetic medium supplemented with pectin as the sole carbon source, and attenuated virulence towards citrus fruits. Introducing the full length of PdpacC into the ΔPdpacC mutant restored all these phenotypes. The expression of the polygalacturonase gene Pdpg2 and pectin lyase gene Pdpnl1 in P. digitatum was upregulated in the wild type strain but not or weakly upregulated in the ΔPdpacC mutant during infection. Disruption of Pdpg2 also resulted in attenuated virulence of P. digitatum towards citrus fruits. Collectively, we conclude that PdPacC plays an important role in pathogenesis of P. digitatum via regulation of the expression of cell wall degradation enzyme genes, such as Pdpg2 and Pdpnl1.This work was supported by the National Foundation of Natural Science of China (31071649), China Agriculture Research System (CARS-27) and the Special Fund for Agro-scientific Research in the Public Interest (201203034).Peer Reviewe

Characterization and disruption of the cipC gene in the ochratoxigenic fungus Aspergillus carbonarius

Aspergillus carbonarius is considered the most important ochratoxin A (OTA) producing fungi among those causing OTA contamination in grapes and grape-derived products. CipC is a small protein with unknown function that was previously found to be highly up-regulated in an OTA producer strain of A. carbonarius in comparison to a non OTA producer strain. In this study, cipC was characterized and disrupted via Agrobacterium tumefaciens-mediated transformation in an ochratoxigenic A. carbonarius strain in order to study whether this gene has a role in OTA production. Sequence analysis indicated that the promoter region of cipC contains putative binding sites for transcription factors that regulate the utilization of nutrients, the stress response and detoxification processes, all factors that can influence mycotoxin biosynthesis. Although the {increment}. cipC mutant grew similarly to the wild type strain, the null mutant showed a much higher OTA production. Moreover, when A. carbonarius was grown under the oxidative stress conditions imposed by the presence of hydrogen peroxide, cipC gene expression was up-regulated. These results indicate that cipC is not directly involved in OTA biosynthesis, but sequence analysis of the A. carbonarius cipC gene promoter and the phenotype of the δ cipC disrupted mutant suggests that CipC could be a stress response protein that would be up-regulated concomitantly with OTA production. © 2013 Elsevier Ltd.A. Crespo-Sempere was recipient of a FPI fellowship from the Spanish Government. This research was supported by grants AGL2005-00707 and AGL2008-04828-C03-02 from the Spanish Government.Peer Reviewe

Development of a green fluorescent tagged strain of Aspergillus carbonarius to monitor fungal colonization in grapes

An enhanced green fluorescent protein has been used to tag an OTA-producing strain of Aspergillus carbonarius (W04-40) isolated from naturally infected grape berries. Transformation of the fungus was mediated by Agrobacterium tumefaciens. The most efficient transformation occurred when the co-cultivation was done with 104 conidia due to higher frequency of resistance colonies (894 per 104 conidia) and lower background obtained. To confirm the presence of the hph gene in hygromycin resistant colonies, 20 putative transformants were screened by PCR analysis. The hph gene was identified in all the transformants. Variation on the expression levels of the eGFP was detected among the transformants and 50% of them appeared bright green fluorescent under the microscope. Microscopic analysis of all the bright fluorescent transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the conidia. eGFP expression in A. carbonarius was shown to be stable in all transformants. Confocal Laser scanning microscopy images of grape berries infected with the eGFP transformant demonstrated fungal penetration into the berry tissues. OTA production was importantly increased in the eGFP transformant in comparison with the wild type strain and pathogenicity on grape berries was slightly decreased after four days of inoculation. However, no differences in virulence were found after seven days of inoculation, thus allowing utilization of this eGFP mutant for in situ analysis of A. carbonarius infection of grape berries. To our knowledge, this is the first report describing the construction of a GFP-tagged strain belonging to Aspergillus section Nigri for monitoring Aspergillus rot on grape berries.This research was supported by AGL2005-00707/ALI and AGL2008-04828-C03-02 grants from the Spanish Government.Peer Reviewe

A transcriptomic approach highlights induction of secondary metabolism in citrus fruit in response to Penicillium digitatum infection

<p>Abstract</p> <p>Background</p> <p>Postharvest losses of citrus fruit due to green mold decay, caused by the fungus <it>Penicillium digitaum</it>, have a considerable economic impact. However, little is known about the molecular processes underlying the response of citrus fruit to <it>P. digitatum</it>.</p> <p>Results</p> <p>Here we describe the construction of a subtracted cDNA library enriched in citrus genes preferentially expressed in response to pathogen infection followed by cDNA macroarray hybridization to investigate gene expression during the early stages of colonization of the fruit's peel by <it>P. digitatum</it>. Sequence annotation of clones from the subtracted cDNA library revealed that induction of secondary and amino acid metabolisms constitutes the major response of citrus fruits to <it>P. digitatum </it>infection. Macroarray hybridization analysis was conducted with RNA from either control, wounded, ethylene treated or <it>P. digitatum </it>infected fruit. Results indicate an extensive overlap in the response triggered by the three treatments, but also demonstrated specific patterns of gene expression in response to each stimulus. Collectively our data indicate a significant presence of isoprenoid, alkaloid and phenylpropanoid biosynthetic genes in the transcriptomic response of citrus fruits to <it>P. digitatum </it>infection. About half of the genes that are up-regulated in response to pathogen infection are also induced by ethylene, but many examples of ethylene-independent gene regulation were also found. Two notable examples of this regulation pattern are the genes showing homology to a caffeine synthase and a berberine bridge enzyme, two proteins involved in alkaloid biosynthesis, which are among the most induced genes upon <it>P. digitatum </it>infection but are not responsive to ethylene.</p> <p>Conclusions</p> <p>This study provided the first global picture of the gene expression changes in citrus fruit in response to <it>P. digitatum </it>infection, emphasizing differences and commonalities with those triggered by wounding or exogenous ethylene treatment. Interpretation of the differentially expressed genes revealed that metabolism is redirected to the synthesis of isoprenes, alkaloids and phenylpropanoids.</p

Antibodies against Chlamydia pneumoniae in stable angina and interleukin-6 levels

Producción CientíficaInflammation is a key mechanism in atherogenesis and the rapid progression of coronary artery disease. Tissue lesion occasions the release of chemical mediators, cytokines, accompanied by an increase in the blood concentrations of acute phase reactants, such as fibrinogen, C-reactive protein, serum amyloid A protein, sialic acid and ceruloplasmin and a reduction of those of albumin. It has been observed that these proteins are higher in patients with is-chemic heart disease and, furthermore, who have a higher tendency to present adverse cardiovascular incidents [1]. On the other hand, the inflammation appears to be directly linked to the ‘vulnerability’ or ‘instability’ of the atheromatous plaques that pre-dispose to disruption and acute coronary incidents. The inflammatory mechanism, therefore, can repre-sent the final common connection channel of chronic infection between atherogenesis and the clinical manifestations of coronary artery disease [2]

Antibodies against Chlamydia pneumoniae and their relation to lymphocyte population levels

Producción CientíficaChlamydia pneumoniae infection has long been suspected as a possible cause of atherosclerosis and has been frequently detected in atheromatous plaques of the coronary arteries. Nevertheless, its distribution is not correlated to the severity or extent of the disease, but it would support the hypothesis that the organism may be an active factor in the pathogenesis of atherosclerosis. A group of patients with stable angina were examined as to whether or not the positivity of antibodies against Chlamydia pneumoniae modified cellular populations as mechanisms responsible for the alterations of inflammatory response. We concluded that the presence of IgG anti-C. pneumoniae antibodies do not participate in the activation of inflammatory mechanisms that may intervene in the genesis of atherosclerosis in patients with stable angina

Molecular aspects in pathogen-fruit interactions: Virulence and resistance

Fruit losses during postharvest storage and handling due to pathogen infections are one of the major problems in the global food chain supply. The application of chemical fungicides to control diseases is currently limited by legislation in some countries and also raises concerns about food and environmental safety. Exploring molecular aspects of pathogen-fruit interactions therefore has biological and economic significance as a means to help develop rational alternatives for disease control. In this review we present the current knowledge of molecular aspects in pathogen-fruit interactions, addressing the following topics: the application of new “omics” technologies for studying these interactions; the molecular mechanisms of fungal pathogen attack; the regulation of virulence by exogenous factors; and, finally, fruit defense mechanisms.Work in Tian’s lab has been funded by the National Natural Science Foundation of China (31530057; 31371863). Work in the LGC lab has been funded by the Spanish Ministry of Economy and Competitiveness (AGL2011-30519-C03-01 and AGL2014-55802-R) and the Generalitat Valenciana (PrometeoII/2014/027). Work in the IRTA lab has been funded by the Spanish Ministry of Economy and Competitiveness by three national projects AGL2008-04828-C01/AGR, AGL2011-30519-C03/AGR and AGL2014-55287-C02.Peer reviewe

Citrus phenylpropanoids and defence against pathogens. Part I: Metabolic profiling in elicited fruits

Penicillium spp. are among the major postharvest pathogens of citrus fruit. Induction of natural resistance in fruits constitutes one of the alternatives to chemical fungicides. Here, we investigated the involvement of the phenylpropanoid pathway in the induction of resistance in Navelate oranges by examining changes in the metabolic profile of upon eliciting citrus fruits. By using both HPLC-PDA-FD and HPLC-PDA-QTOF-MS allowed the identification of several compounds that seem to be relevant for induced resistance. In elicited fruits, a greater diversity of phenolic compounds was observed in the flavedo (outer coloured part of the peel) when compared to the albedo (inner white part). Moreover, only small changes were detected in the most abundant citrus flavonoids. The coumarin scoparone was among the compounds with the highest induction upon elicitation. Two other highly induced compounds were identified as citrusnin A and drupanin aldehyde. All three compounds are known to exert antimicrobial activity. Our results suggest that phenylpropanoids and their derivatives play an important role in the induction of resistance in citrus fruit.This work was supported by Research Grants AGL2008-04828-C03-02, AGL2009-11969 and CONSOLIDER FUNC-FOOD from the Spanish Ministry of Science and Technology, and PROMETEO/2010/010 from the Generalitat Valenciana.Peer Reviewe