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Sugars and pH: A clue to the regulation of fungal cell wall-degrading enzymes in plants
Physiological and Molecular Plant Pathology 65 (2004) 271–275 www.elsevier.com/locate/pmpp
Mini-review
Sugars and pH: A clue to the regulation of fungal cell wall-degrading enzymes in plants K. Akimitsua,b,*, A. Isshikia, K. Ohtanib, H. Yamamotoa, D. Eshelc, D. Pruskyc a
Laboratory of Plant Pathology, Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan b PRESTO, Japan Science and Technology Agency, Tokyo, Japan c Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel
Abstract In recent years, substantial progress has been made in understanding the role of cell wall-degrading enzymes in virulence of fungal pathogens using genetic tools. A homologous recombination-mediated targeted gene disruption of a single pectolytic gene decreased virulence in multiple fungal pathogens. Furthermore, differential regulation of a gene that encodes a cell wall-degrading enzyme during infection under different host environments has recently been examined, and host pH and sugars are involved in the regulation of the polygalacturonase gene. We will provide a brief overview of the biology of the macerating diseases caused by phytopathogenic Alternaria pathogens in particular hosts, followed by a description of possible mechanisms controlling the differential expression of genes encoding cell wall-degrading enzymes in the infected host. q 2005 Elsevier Ltd. All rights reserved. Keywords: Cell wall-degrading enzymes; Alternaria citri; Alternaria alternata; Citrus; Persimmon fruit; Endopolygalacturonase; pH; Carbon catabolite repression
1. General Introduction Plant cell wall polymers have a dual function as a potential barrier to the penetration and spread of phytopathogens, but at the same time, as substrates for extracellular enzymes secreted by bacterial and fungal pathogens. The role of these cell wall-degrading enzymes (CWDE) in many aspects of pathogenicity, including penetration, tissue maceration, nutrient acquisition, symptom expression, and plant defense induction has been studied [9,10,42]. Although CWDE production has been correlated with pathogenicity and CWDE activity and its gene expression have been detected in infected tissues, no direct involvement of CWDEs has been shown until recently with the use of genetic tools. For example, a homologous recombination-mediated targeted disruption of a single gene of polygalacturonase (endoPG: poly(1,4-aD-galacturonide) glycanohydrolase, E.C. 3.2.1.15) from * Corresponding author. Tel.: C81 87 891 3131; fax: C81 87 891 3021. E-mail address: [email protected] (K. Akimitsu).
0885-5765/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2005.03.001
Aspergillus flavus on cotton bolls, Botrytis cinerea on tomato and Alternaria citri on citrus [18,38,39] decreased virulence. Also in Colletotrichum gloeosporioides attacking avocado fruits, a single disruption of the pectate lyase (PL) gene reduced virulence of the mutant [47]. In other cases, as in Nectria hematococca on pea, the disruption of a single pectate lyase gene did not reduce virulence; two PL genes were disrupted to reduce virulence [34]. Beside these disruptions that reduced virulence, virulence was not decreased in many other disruption studies on endoPG, exoPG, xylanases, xylosidase, protease, and glucanases of Cochliobolus carbonum [2,17,25,35–37,44]. These results exemplify the challenge for evaluating the single effect of a CWDE because of its dual function during pathogenicity, the possibility of retaining residual enzyme activity, or other pathogenicity factors that may compensate for the lack of a single enzyme in the disruption mutants. Scott-Craig et al. [37] disrupted two genes, encoding endoPG and exoPG, and found decreased growth on pectin without having any effect on virulence, may indicate more role of CWDE for growth and nutrient acquisition in C. carbonum. Furthermore, a decrease in virulence by disruption of CWDE was observed more frequently in pathogens causing rot symptom than in
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those causing other symptoms, such as leaf spot, because the role of CWDEs in virulence is likely to macerate host tissues. When we evaluated the role of endoPG in the pathogenicity of two different but morphologically indistinguishable citrus pathogens of Alternaria spp., we found that one (A. citri) causes rot symptoms by macerating tissues, and the other (Alternaria alternata rough lemon pathotype) causes necrotic spots by secreting a hostselective toxin, the phenotypes of the endoPG-disrupted mutants were completely different [18]. Although the genes and the activities of the encoded endoPGs of both pathogens were similar (99.6% nucleotide identity) and have similar biochemical properties, the endoPG mutant of A. citri had reduced ability to cause black rot symptoms on citrus, but the endoPG-disrupted mutant of A. alternata had no reduction in pathogenicity [18]. The results indicate that CWDEs can play different roles in the pathogenicity of similar sp., and the role of a CWDE may be indicated by the type of disease symptoms but not the identity of the fungus [18]. Two possibilities may explain why disruption of endoPG in A. citri reduced virulence so dramatically. The mutant barely could grow on pectin or polygalacturonic acid (PGA) as the sole carbon source, indicating that the disrupted endoPG was essential for degradation of pectic components. Other pectic enzymes, including the exoPG, were not sufficient for nutrient acquisition. The disrupted endoPG gene had a single copy in the A. citri strain used that is an advantage for evaluation of the role of endoPG in this pathogen [18]. Second, A. citri attacks the albedo that is very rich in pectin, the degradation of which is probably very important for successful fruit colonization. In leaves, which have far less pectin, symptoms are rarely found [6,7]. Thus, disrupting the endoPG gene and reducing or eliminating the carbon source may inhibit fungal growth in the fruit. On the contrary, the pathogenicity of an endoPG-disrupted mutant of A. alternata rough lemon pathotype, a necrotrophic pathogen producing host-selective ACR-toxin [1,16,22,26] did not obviously change because of the reduced contribution of CWDE in symptom development by this strain [18]. Because the highly toxic ACR-toxin can induce necrotic spots, CWDE probably contributes less to symptom development [18]. Walton’s group [41] took a different approach to evaluate the role of multiple CWDEs in pathogenesis. With the disruption of ccSNF1, a gene required for expression of catabolite-repressed genes under glucose limitation in the maize pathogen C. carbonum, genes encoding CWDEs were down-regulated, and virulence of the mutant decreased [41]. These results indicate that gene regulation of CWDEs is modulated by the signal transduction processes controlled by ccSNF1, and degradation of polymers of the plant cell wall are essential for pathogenesis on maize by C. carbonum [41].
2. Regulation of CWDE during pathogenicity Although several lines of evidence implicate CWDEs in the virulence of fungal, the mechanism for the regulation of gene expression for these enzymes in different tissues of host plants during different phases of infection is still ambiguous. An increase or decrease in CWDE gene expression, equal to CWDE activity, might be affected not only by pathogen factors but also by host plant factors during infection [9,10]. One approach to analyze differential regulation of the CWDE gene during infection is to use RT-PCR, western blot, or promoter analysis using green fluorescent protein (GFP) as a reporter [12,18,20,27,32,46]. Results with this methodology suggested that host factors and environmental conditions may be involved in the regulation of CWDE in phytopathogenic Alternaria pathogens of black rot of citrus and black spot of persimmon fruit as described in a later section. A. citri Ellis and N. Pierce causes Alternaria black rot, a postharvest fruit disease of a broad range of citrus cultivars [6,7]. The disease, reported as early as the 1900s [4,5,30,33], produces internal decay in the central columella area of the fruit of all commercial citrus [7]. A small brown to black spot sometimes appears on the stylar end of the fruit, but unlike other citrus pathogens that cause rot symptoms, such as Penicillium digitatum and P. italicum, this pathogen usually does not colonize the external surface of the infected fruits [6]. As described earlier, we demonstrated that the pathogenicity of this pathogen depends upon production of an extracellular endoPG that can degrade pectic polymers in cell walls during the infection stage. The purified endoPG has a molecular mass of 60 kDa, optimum pH of approximately 5.0, and a Km of 0.15 mg PGA per ml. After cloning the endoPG gene from A. citri [18,19] and disrupting it, soft rot symptoms were reduced by 85% as a result of the inhibition of penetration and maceration of citrus tissue [18,20]. In vitro radial growth of the endoPG-disrupted mutant was not significantly reduced when glucose was the sole carbon source, but it was reduced severely on pectin, PGA, or purified citrus cell walls [18].
3. Role of endoPG during different phases of fungal infection Some biotrophic pathogens grow and develop in the middle lamella between the cuticle and outer epidermal cell wall [9,10,20], which is mainly composed of pectin [8,11]. EndoPGs of these biotrophs often have large molecular weights and are bound to the fungal wall, preventing diffusion through the wall matrix, resulting in localized degradation of the plant cell wall to minimize host damage and thereby establish a biotrophic relationship between the pathogens and their hosts [9,10]. Although A. citri is
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considered to be a necrotrophic fungus and the endoPG from A. citri has no special characteristics of biotrophic endoPGs (such as being large or bound to the fungal wall), this pathogen was thought to colonize pectin-rich tissue parts during infection, as do biotrophic fungi [6,7]. When GFP gene was introduced into both wild type A. citri and its endoPG-disrupted mutant [20], it was observed that hyphae of the wild type but not those of disrupted mutant grew through the central axis from the pedicel in the peel with its pectin-rich tissues. However, the hyphae of both the WT and the disrupted mutant spread equally within the juice sac area of the fruit [20]. These results indicate that endoPG is essential for penetration and rotting during disease development, but endoPG is not important for fungal colonization in the juice sac area [18,20].
4. Regulation of endoPG by carbon catabolite repression When the GFP gene was fused to the endoPG promoter, the regulation of endoPG expression could be detected already in germinating spores on agar medium containing pectin or PGA as a sole carbon source. However, no fluorescence was detected when the fungus was grown with cellulose as either the sole or additional carbon source [20,27]. Nor was green fluorescence induced on pectin media containing 1% (w/v) glucose or higher, indicating that the endoPG gene expression is regulated under carbon catabolite repression, when a range of genes are repressed by the presence of readily utilizable carbon sources such as glucose. Carbon catabolite repression of fungal PGs has been reported for Fusarium oxysporum f. sp. lycopersici [28], Aspergillus niger [23], Aspergillus nidulans [3], C. carbonum race 1 [43] and Penicillium expansum [29]. Similar to other fungal PGs, accumulation of endoPG transcripts and their activities were also repressed in A. citri when 1 or 2% (w/v) glucose was added to the pectin medium [20,27]. To investigate if the differential development of A. citri in the pectin part of the peel tissue and in the juice sacs is the result of catabolic repression [20,27] spore suspensions of the transformant were spread on pectin or PGA plates containing 2% (w/v) fructose or sucrose (the major sugars in citrus juice at concentrations up to 5–10% [w/v]) [24,40], and no GFP expression was detected on plates with 2% (w/v) fructose, sucrose or glucose [20,27]. This simple experiment suggests that the concentration of these sugars in citrus juice is high enough to repress endoPG gene expression in the juice sac area. The GFP gene was further employed as a reporter gene to define the region comprising the endoPG promoter of A. citri with the use of several deletions in the 813 bp upstream from the translation start site [27]. This 813-bp sequence contains putative binding sites for catabolite repressive element A (CreA), a cis-acting zinc finger repressor involved in carbon catabolite repression [20,27].
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For that study we constructed a fusion of CreA-binding site-deleted endoPG promoters with the GFP reporter gene and transformed the wild-type black rot fungus. The construct PGPDL4 had a K401 to K813 deletion with both substrate induction and catabolite repression, while PGPDL5 with substrate induction had an additional deletion from K1 to K84, including one putative CreAbinding site, which resulted in the loss of catabolite repression [27]. Green fluorescence of PGPDL4 was induced by pectin in the peel but repressed completely in the juice sac area of citrus fruit. However, PGPDL5 fluoresced green in both the peel and juice sac area, again indicating that the repression of A. citri endoPG gene in the juice sac area is likely by carbon catabolite repression [27]. A gene encoding CreA was cloned, and research on the role of CreA in catabolite repression of A. citri is currently progressing (Katoh, Ohtani and Akimitsu, unpublished).
5. Regulation of pectinase expression by pH It was recently reported that ambient alkalinization is a possible factor modulating pathogenicity in Alternaria [31]. The affected cell tissue shows enhanced elicitation of the main virulence factors that are secreted by the pathogen. This behavior enables a pathogen to select the specific virulence factor needed for a particular host, with no need to express its entire battery of genes. This flexibility, in both the deployment of potential virulence factors and the active adjustment of the ambient environment to provide the optimal conditions for the virulence factors, demonstrates the complexity for modulation pathogenicity by pathogens as Alternaria. Ambient alkalization in Alternaria during fruit colonization is achieved by the secretion of ammonia by the pathogen [31], which might be activated by fungal proteases. Subsequent determination of amino acids is responsible for the alkalization [21]. In the case of A. alternata, ammonia accumulation (a threefold to tenfold increase) and a pH increase (0.2–2.4 pH units) have been detected in several hosts [14,31]. Tissue pH is an important parameter in aqueous environments, because it affects the activities of individual enzymes, which in turn may determine virulence. Optima pH required for their pathogenicities of A. alternata that attack distinct hosts are different. For example, Alternaria attacks citrus with a low pH of 3.0–3.5 and persimmons having a pH of 5.8–6.0. We envision that in polyphagous pathogens, different genes encoding for pectolytic enzymes might be activated at different environmental pH levels. This could require the pathogen to have a battery of different enzymes as virulence factors for a specific environment. EndoPG is essential for citrus black rot caused by A. citri [18], while endoglucanase (EG) of A. alternata may affect virulence in other fruits as persimmons [13,14]. Such differential enzymatic activation
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may explain cases in which inactivation of a single pectolytic gene had no discernible effect on the pathogenicity of the fungus on its host plant [15,37]. A. alternata EG AaK1 expression, EG production, and virulence were also repressed by glucose and affected by pH alkalinization [13]. Glucose repression was, however, accompanied by a significant decrease in pH from 3.8 to 2.5. Repression of virulence by glucose is probably due to the acidification of the fruit tissue, because buffer restored virulence. However, acidification of the growth media by A. alternata in the presence of glucose raises the question of whether the gene repression caused by glucose is caused by a decrease in environmental pH created during glucose metabolism. When A. alternata was grown in the presence of cell walls and glucose at pH 6.0, transcript expression, EG production and virulence were significantly higher. The results suggest that ambient pH depressed the glucose repression in the modulation of the AaK1 expression. This is supported by early findings on B. cinerea in which Bcpg gene expression in the presence of glucose was also altered by pH levels [45]. These phenomena highlight the influence of new environmental conditions created by the pathogen on the outcome of the host interactions.
6. Conclusion Based on the results in studies on Alternaria black rot, different pathogenicity factors either toxins and pectolytic enzymes are differential expressed during the penetration and symptoms development of Alternaria in different fruit tissue. EndoPG is not necessary for hyphal growth and colonization of the juice sac, and it is differentially regulated in different areas of the citrus fruit during disease development. However, multiple factors in host tissues, such as carbon source and pH, at each developmental stage influence the complex regulation of CWDE genes and the biochemical specialization of the CWDE. A number of regulators for such signals, CreA and PacC (a pH signaling transcription factor), are also likely to be involved in CWDE gene expression, and hence relate directly to the virulence of phytopathogens, which depend on CWDEs for their survival.
Acknowledgements These studies were supported in part by grants in priority area A from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K.A.), and Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency (to K.A.).
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Mini-review
Sugars and pH: A clue to the regulation of fungal cell wall-degrading enzymes in plants K. Akimitsua,b,*, A. Isshikia, K. Ohtanib, H. Yamamotoa, D. Eshelc, D. Pruskyc a
Laboratory of Plant Pathology, Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan b PRESTO, Japan Science and Technology Agency, Tokyo, Japan c Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel
Abstract In recent years, substantial progress has been made in understanding the role of cell wall-degrading enzymes in virulence of fungal pathogens using genetic tools. A homologous recombination-mediated targeted gene disruption of a single pectolytic gene decreased virulence in multiple fungal pathogens. Furthermore, differential regulation of a gene that encodes a cell wall-degrading enzyme during infection under different host environments has recently been examined, and host pH and sugars are involved in the regulation of the polygalacturonase gene. We will provide a brief overview of the biology of the macerating diseases caused by phytopathogenic Alternaria pathogens in particular hosts, followed by a description of possible mechanisms controlling the differential expression of genes encoding cell wall-degrading enzymes in the infected host. q 2005 Elsevier Ltd. All rights reserved. Keywords: Cell wall-degrading enzymes; Alternaria citri; Alternaria alternata; Citrus; Persimmon fruit; Endopolygalacturonase; pH; Carbon catabolite repression
1. General Introduction Plant cell wall polymers have a dual function as a potential barrier to the penetration and spread of phytopathogens, but at the same time, as substrates for extracellular enzymes secreted by bacterial and fungal pathogens. The role of these cell wall-degrading enzymes (CWDE) in many aspects of pathogenicity, including penetration, tissue maceration, nutrient acquisition, symptom expression, and plant defense induction has been studied [9,10,42]. Although CWDE production has been correlated with pathogenicity and CWDE activity and its gene expression have been detected in infected tissues, no direct involvement of CWDEs has been shown until recently with the use of genetic tools. For example, a homologous recombination-mediated targeted disruption of a single gene of polygalacturonase (endoPG: poly(1,4-aD-galacturonide) glycanohydrolase, E.C. 3.2.1.15) from * Corresponding author. Tel.: C81 87 891 3131; fax: C81 87 891 3021. E-mail address: [email protected] (K. Akimitsu).
0885-5765/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2005.03.001
Aspergillus flavus on cotton bolls, Botrytis cinerea on tomato and Alternaria citri on citrus [18,38,39] decreased virulence. Also in Colletotrichum gloeosporioides attacking avocado fruits, a single disruption of the pectate lyase (PL) gene reduced virulence of the mutant [47]. In other cases, as in Nectria hematococca on pea, the disruption of a single pectate lyase gene did not reduce virulence; two PL genes were disrupted to reduce virulence [34]. Beside these disruptions that reduced virulence, virulence was not decreased in many other disruption studies on endoPG, exoPG, xylanases, xylosidase, protease, and glucanases of Cochliobolus carbonum [2,17,25,35–37,44]. These results exemplify the challenge for evaluating the single effect of a CWDE because of its dual function during pathogenicity, the possibility of retaining residual enzyme activity, or other pathogenicity factors that may compensate for the lack of a single enzyme in the disruption mutants. Scott-Craig et al. [37] disrupted two genes, encoding endoPG and exoPG, and found decreased growth on pectin without having any effect on virulence, may indicate more role of CWDE for growth and nutrient acquisition in C. carbonum. Furthermore, a decrease in virulence by disruption of CWDE was observed more frequently in pathogens causing rot symptom than in
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those causing other symptoms, such as leaf spot, because the role of CWDEs in virulence is likely to macerate host tissues. When we evaluated the role of endoPG in the pathogenicity of two different but morphologically indistinguishable citrus pathogens of Alternaria spp., we found that one (A. citri) causes rot symptoms by macerating tissues, and the other (Alternaria alternata rough lemon pathotype) causes necrotic spots by secreting a hostselective toxin, the phenotypes of the endoPG-disrupted mutants were completely different [18]. Although the genes and the activities of the encoded endoPGs of both pathogens were similar (99.6% nucleotide identity) and have similar biochemical properties, the endoPG mutant of A. citri had reduced ability to cause black rot symptoms on citrus, but the endoPG-disrupted mutant of A. alternata had no reduction in pathogenicity [18]. The results indicate that CWDEs can play different roles in the pathogenicity of similar sp., and the role of a CWDE may be indicated by the type of disease symptoms but not the identity of the fungus [18]. Two possibilities may explain why disruption of endoPG in A. citri reduced virulence so dramatically. The mutant barely could grow on pectin or polygalacturonic acid (PGA) as the sole carbon source, indicating that the disrupted endoPG was essential for degradation of pectic components. Other pectic enzymes, including the exoPG, were not sufficient for nutrient acquisition. The disrupted endoPG gene had a single copy in the A. citri strain used that is an advantage for evaluation of the role of endoPG in this pathogen [18]. Second, A. citri attacks the albedo that is very rich in pectin, the degradation of which is probably very important for successful fruit colonization. In leaves, which have far less pectin, symptoms are rarely found [6,7]. Thus, disrupting the endoPG gene and reducing or eliminating the carbon source may inhibit fungal growth in the fruit. On the contrary, the pathogenicity of an endoPG-disrupted mutant of A. alternata rough lemon pathotype, a necrotrophic pathogen producing host-selective ACR-toxin [1,16,22,26] did not obviously change because of the reduced contribution of CWDE in symptom development by this strain [18]. Because the highly toxic ACR-toxin can induce necrotic spots, CWDE probably contributes less to symptom development [18]. Walton’s group [41] took a different approach to evaluate the role of multiple CWDEs in pathogenesis. With the disruption of ccSNF1, a gene required for expression of catabolite-repressed genes under glucose limitation in the maize pathogen C. carbonum, genes encoding CWDEs were down-regulated, and virulence of the mutant decreased [41]. These results indicate that gene regulation of CWDEs is modulated by the signal transduction processes controlled by ccSNF1, and degradation of polymers of the plant cell wall are essential for pathogenesis on maize by C. carbonum [41].
2. Regulation of CWDE during pathogenicity Although several lines of evidence implicate CWDEs in the virulence of fungal, the mechanism for the regulation of gene expression for these enzymes in different tissues of host plants during different phases of infection is still ambiguous. An increase or decrease in CWDE gene expression, equal to CWDE activity, might be affected not only by pathogen factors but also by host plant factors during infection [9,10]. One approach to analyze differential regulation of the CWDE gene during infection is to use RT-PCR, western blot, or promoter analysis using green fluorescent protein (GFP) as a reporter [12,18,20,27,32,46]. Results with this methodology suggested that host factors and environmental conditions may be involved in the regulation of CWDE in phytopathogenic Alternaria pathogens of black rot of citrus and black spot of persimmon fruit as described in a later section. A. citri Ellis and N. Pierce causes Alternaria black rot, a postharvest fruit disease of a broad range of citrus cultivars [6,7]. The disease, reported as early as the 1900s [4,5,30,33], produces internal decay in the central columella area of the fruit of all commercial citrus [7]. A small brown to black spot sometimes appears on the stylar end of the fruit, but unlike other citrus pathogens that cause rot symptoms, such as Penicillium digitatum and P. italicum, this pathogen usually does not colonize the external surface of the infected fruits [6]. As described earlier, we demonstrated that the pathogenicity of this pathogen depends upon production of an extracellular endoPG that can degrade pectic polymers in cell walls during the infection stage. The purified endoPG has a molecular mass of 60 kDa, optimum pH of approximately 5.0, and a Km of 0.15 mg PGA per ml. After cloning the endoPG gene from A. citri [18,19] and disrupting it, soft rot symptoms were reduced by 85% as a result of the inhibition of penetration and maceration of citrus tissue [18,20]. In vitro radial growth of the endoPG-disrupted mutant was not significantly reduced when glucose was the sole carbon source, but it was reduced severely on pectin, PGA, or purified citrus cell walls [18].
3. Role of endoPG during different phases of fungal infection Some biotrophic pathogens grow and develop in the middle lamella between the cuticle and outer epidermal cell wall [9,10,20], which is mainly composed of pectin [8,11]. EndoPGs of these biotrophs often have large molecular weights and are bound to the fungal wall, preventing diffusion through the wall matrix, resulting in localized degradation of the plant cell wall to minimize host damage and thereby establish a biotrophic relationship between the pathogens and their hosts [9,10]. Although A. citri is
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considered to be a necrotrophic fungus and the endoPG from A. citri has no special characteristics of biotrophic endoPGs (such as being large or bound to the fungal wall), this pathogen was thought to colonize pectin-rich tissue parts during infection, as do biotrophic fungi [6,7]. When GFP gene was introduced into both wild type A. citri and its endoPG-disrupted mutant [20], it was observed that hyphae of the wild type but not those of disrupted mutant grew through the central axis from the pedicel in the peel with its pectin-rich tissues. However, the hyphae of both the WT and the disrupted mutant spread equally within the juice sac area of the fruit [20]. These results indicate that endoPG is essential for penetration and rotting during disease development, but endoPG is not important for fungal colonization in the juice sac area [18,20].
4. Regulation of endoPG by carbon catabolite repression When the GFP gene was fused to the endoPG promoter, the regulation of endoPG expression could be detected already in germinating spores on agar medium containing pectin or PGA as a sole carbon source. However, no fluorescence was detected when the fungus was grown with cellulose as either the sole or additional carbon source [20,27]. Nor was green fluorescence induced on pectin media containing 1% (w/v) glucose or higher, indicating that the endoPG gene expression is regulated under carbon catabolite repression, when a range of genes are repressed by the presence of readily utilizable carbon sources such as glucose. Carbon catabolite repression of fungal PGs has been reported for Fusarium oxysporum f. sp. lycopersici [28], Aspergillus niger [23], Aspergillus nidulans [3], C. carbonum race 1 [43] and Penicillium expansum [29]. Similar to other fungal PGs, accumulation of endoPG transcripts and their activities were also repressed in A. citri when 1 or 2% (w/v) glucose was added to the pectin medium [20,27]. To investigate if the differential development of A. citri in the pectin part of the peel tissue and in the juice sacs is the result of catabolic repression [20,27] spore suspensions of the transformant were spread on pectin or PGA plates containing 2% (w/v) fructose or sucrose (the major sugars in citrus juice at concentrations up to 5–10% [w/v]) [24,40], and no GFP expression was detected on plates with 2% (w/v) fructose, sucrose or glucose [20,27]. This simple experiment suggests that the concentration of these sugars in citrus juice is high enough to repress endoPG gene expression in the juice sac area. The GFP gene was further employed as a reporter gene to define the region comprising the endoPG promoter of A. citri with the use of several deletions in the 813 bp upstream from the translation start site [27]. This 813-bp sequence contains putative binding sites for catabolite repressive element A (CreA), a cis-acting zinc finger repressor involved in carbon catabolite repression [20,27].
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For that study we constructed a fusion of CreA-binding site-deleted endoPG promoters with the GFP reporter gene and transformed the wild-type black rot fungus. The construct PGPDL4 had a K401 to K813 deletion with both substrate induction and catabolite repression, while PGPDL5 with substrate induction had an additional deletion from K1 to K84, including one putative CreAbinding site, which resulted in the loss of catabolite repression [27]. Green fluorescence of PGPDL4 was induced by pectin in the peel but repressed completely in the juice sac area of citrus fruit. However, PGPDL5 fluoresced green in both the peel and juice sac area, again indicating that the repression of A. citri endoPG gene in the juice sac area is likely by carbon catabolite repression [27]. A gene encoding CreA was cloned, and research on the role of CreA in catabolite repression of A. citri is currently progressing (Katoh, Ohtani and Akimitsu, unpublished).
5. Regulation of pectinase expression by pH It was recently reported that ambient alkalinization is a possible factor modulating pathogenicity in Alternaria [31]. The affected cell tissue shows enhanced elicitation of the main virulence factors that are secreted by the pathogen. This behavior enables a pathogen to select the specific virulence factor needed for a particular host, with no need to express its entire battery of genes. This flexibility, in both the deployment of potential virulence factors and the active adjustment of the ambient environment to provide the optimal conditions for the virulence factors, demonstrates the complexity for modulation pathogenicity by pathogens as Alternaria. Ambient alkalization in Alternaria during fruit colonization is achieved by the secretion of ammonia by the pathogen [31], which might be activated by fungal proteases. Subsequent determination of amino acids is responsible for the alkalization [21]. In the case of A. alternata, ammonia accumulation (a threefold to tenfold increase) and a pH increase (0.2–2.4 pH units) have been detected in several hosts [14,31]. Tissue pH is an important parameter in aqueous environments, because it affects the activities of individual enzymes, which in turn may determine virulence. Optima pH required for their pathogenicities of A. alternata that attack distinct hosts are different. For example, Alternaria attacks citrus with a low pH of 3.0–3.5 and persimmons having a pH of 5.8–6.0. We envision that in polyphagous pathogens, different genes encoding for pectolytic enzymes might be activated at different environmental pH levels. This could require the pathogen to have a battery of different enzymes as virulence factors for a specific environment. EndoPG is essential for citrus black rot caused by A. citri [18], while endoglucanase (EG) of A. alternata may affect virulence in other fruits as persimmons [13,14]. Such differential enzymatic activation
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may explain cases in which inactivation of a single pectolytic gene had no discernible effect on the pathogenicity of the fungus on its host plant [15,37]. A. alternata EG AaK1 expression, EG production, and virulence were also repressed by glucose and affected by pH alkalinization [13]. Glucose repression was, however, accompanied by a significant decrease in pH from 3.8 to 2.5. Repression of virulence by glucose is probably due to the acidification of the fruit tissue, because buffer restored virulence. However, acidification of the growth media by A. alternata in the presence of glucose raises the question of whether the gene repression caused by glucose is caused by a decrease in environmental pH created during glucose metabolism. When A. alternata was grown in the presence of cell walls and glucose at pH 6.0, transcript expression, EG production and virulence were significantly higher. The results suggest that ambient pH depressed the glucose repression in the modulation of the AaK1 expression. This is supported by early findings on B. cinerea in which Bcpg gene expression in the presence of glucose was also altered by pH levels [45]. These phenomena highlight the influence of new environmental conditions created by the pathogen on the outcome of the host interactions.
6. Conclusion Based on the results in studies on Alternaria black rot, different pathogenicity factors either toxins and pectolytic enzymes are differential expressed during the penetration and symptoms development of Alternaria in different fruit tissue. EndoPG is not necessary for hyphal growth and colonization of the juice sac, and it is differentially regulated in different areas of the citrus fruit during disease development. However, multiple factors in host tissues, such as carbon source and pH, at each developmental stage influence the complex regulation of CWDE genes and the biochemical specialization of the CWDE. A number of regulators for such signals, CreA and PacC (a pH signaling transcription factor), are also likely to be involved in CWDE gene expression, and hence relate directly to the virulence of phytopathogens, which depend on CWDEs for their survival.
Acknowledgements These studies were supported in part by grants in priority area A from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K.A.), and Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency (to K.A.).
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