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Applied and Environmental Microbiology, June 2003, p . 3258-3262, Vol . 69, No . 6

External pH and Nitrogen Source Affect Secretion of Pectate Lyase by Colletotrichum gloeosporioides

N . Drori,1,2 H . Kramer-Haimovich,1,2 J . Rollins,3 A . Dinoor,2 Y . Okon,2 O . Pines,4 and D . Prusky1*

Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250,1 Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, Rehovot 76100,2 Department of Molecular Biology, Hebrew University, Medical School, Jerusalem 91120, Israel,4 Department of Plant Pathology, University of Florida, Gainesville, Florida 326113

Received 28 August 2002/ Accepted 6 March 2003


   ABSTRACT

 
Accumulation of ammonia and associated tissue alkalinization predispose fruit to attack by Colletotrichumgloeosporioides. As the external pH increases from 4.0 to 6.0, pectate lyase (PL) and other extracellular proteins are secreted and accumulate . At pH 4.0 neither pelB (encoding PL) transcription nor PL secretion were detected; however, they were detected as the pH increased . Nitrogen assimilation also was required for PL secretion at pH 6.0 . Both inorganic and organic nitrogen sources enhanced PL secretion at pH 6.0, but neither was sufficient for PL secretion at pH 4.0 . Sequence analysis of the 5' upstream region of the pelB promoter revealed nine putative consensus binding sites for the Aspergillus transcription factor PacC . Consistent with this result, the transcript levels of pac1 (the C . gloeosporioides pacC homologue) and pelB increased in parallel as a function of pH . Our results suggest that the ambient pH and the nitrogen source are independent regulatory factors for processes linked to PL secretion and virulence of C . gloeosporioides .


   INTRODUCTION

 
Colletotrichum gloeosporioides is a filamentous ascomycete phytopathogen that attacks the fruits of many hosts before and after harvest . During colonization by the pathogen ammonia accumulates in the host (14), which creates the alkaline pH environment necessary for the activities of some extracellular lyase enzymes, including pectate lyases (PLs) (3, 4, 26, 27) . PL has been implicated as a virulence factor of C . gloeosporioides in avocado fruit (29), and its expression is strongly affected by alkalinization . Thus, environmental conditions that affect PL secretion may be important virulence factors for C . gloeosporioides .

The alkalinization of the tissue and the increased virulence of C . gloeosporioides also may depend on the availability of exogenous nitrogen, which can be converted to NH4+ . The increase in the amount of NH4+ thus increases the external pH, which leads to expression of different genes, including pelB, the gene encoding PL, and other genes encoding secreted pectolytic enzymes . In Aspergillus nidulans and Sclerotinia sclerotiorum an ambient-pH-sensing signal transduction pathway affects expression of genes encoding several secreted and outer-membrane-bound proteins, as well as enzymes that synthesize exportable metabolites (2, 5, 7, 8, 11, 17, 19, 24) . The product of the pacC gene is the terminal component of the pH signaling pathway and the transcription regulator of pH-dependent gene expression (2) . This protein has a zinc finger DNA-binding domain with the core DNA consensus binding site 5'-GCCARG-3' (24) .

In this study our main objective was to determine the importance of the nitrogen source and the external pH in secretion of the virulence factor PL with respect to the ambient pH transcriptional regulator pacC . We hypothesized that nitrogen source availability and the ambient pH are two independent signals for transcriptional regulation of genes required for the disease processes of C . gloeosporioides and possibly other pathogens .


   MATERIALS AND METHODS

 
Strain, medium, and growth conditions.
Single-spore cultures of the Cg-14 isolate of C . gloeosporioides were obtained from a decayed cultivar Fuerte avocado fruit in Israel . This isolate was routinely cultured on M3S agar containing 250 mg of chloramphenicol per liter . The fungus was grown in 40 ml of M3S medium (25) at pH 5.0 (primary culture) and inoculated by using 106 spores/flask . M3S medium contains (per liter) 2.5 g of MgSO4 · 7H2O, 2.7 g of KH2PO4, 1.5 g of Peptone bios D (Biolife, Milan, Italy), 1.5 g of Bacto Yeast Extract (Difco, Sparks, Md.), and 15 g of sucrose . Cultures were incubated at 22°C in a shaking incubator at 150 rpm for 4 days . Each culture was harvested by filtration over a sterile Buchner funnel fitted with filter paper (Whatman, Maidstone, England), and the hyphae remaining on the filter were washed twice with 40 ml of sterile distilled water each time . The washed mycelia were resuspended in 40 ml of fresh medium (secondary medium) containing (per liter) 4 g of K2HPO4, 2 g of MgSO4 · 7H2O, 5 g of KNO3, 0.3 g of CaCl2 · 2H2O, 10 mg of FeCl3, and 10 g of glucose . To obtain initial pH values between 4.0 and 6.0, the culture was buffered with 0.05 M phthalate hydroxide buffer (Sigma, St . Louis, Mo.) . The initial pH for each flask was determined after the medium was autoclaved but prior to inoculation . Experiments were repeated at least three times, and the results of a single representative experiment are presented below . Differences between average values for each treatment did not differ by more than 2 to 3% between experiments .

The pH was measured with a microcombination pH electrode (model 9810BNp; Orion, Beverly, Mass.) in 0.5-ml aliquots obtained at different times after fungal inoculation .

Cloning and sequencing of pac1 from C . gloeosporioides.
DNA fragments corresponding to nucleotides 1195 to 1387 of the Aspergillus nidulans pacC gene (accession no . Z47081) were amplified by performing PCR with C . gloeosporioides genomic DNA and the following primers: PACZNF3 (5'-GTGTGCGAGCGTCACGTAGG-3') and PACZN-R1 (5'-ACATGTTTCAAGTCCTG-3') . The fragments were cloned by using a TOPO TA cloning kit into pCRII (Invitrogen, Carlsbad, Calif.) to generate pCGPAC . Genomic DNA was isolated from the mycelium of C . gloeosporioides by using a DNeasy QIAGEN kit (QIAGEN, Santa Clarita, Calif.) .

Plasmid pCGPAC contained a partial genomic sequence of C . gloeosporioides pac1 and was used as a DNA sequencing template . Both strands of the insert were sequenced by using the T7 and SP6 primers (DNA Sequencing Facility, University of Florida, Gainesville) . Sequence homology analysis was conducted by using the BLAST algorithm . The 197-nucleotide sequence is 81% identical to the A . nidulans pacC sequence . The predicted 64 amino acids encoded are 95% identical to the amino acids in the corresponding A . nidulans PacC sequence .

Detection of PL in liquid medium and fungal hyphae.
Secondary cultures were separated from the medium by filtration, as described above, and the hyphae were washed twice with sterile water, frozen with liquid nitrogen, lyophilized, and stored at -80°C until they were used for RNA or protein extraction . The culture medium filtrate was concentrated with a rotaevaporator (Buchii, Flawil, Switzerland) at 30°C to 5 ml, dialyzed by using SnakeSkin pleated dialysis tubing (molecular weight cutoff, 10,000; Pierce, Rockford, Ill.) for 24 h against 5 liters of 50 mM Tris-HCl (pH 8.5), and then reconcentrated to 1 ml, lyophilized, and resuspended in 150 µl of sterile water . Protein samples were quantified with the Bio-Rad Laboratories (Hercules, Calif.) protein assay by using bovine serum albumin (Sigma) as a standard . Mycelial (nonsecreted) proteins of C . gloeosporioides were extracted from 25 mg of dried hyphae in the presence of 1 ml of 0.5 M Tricine buffer (pH 8.3) containing 0.1 M cetyltrimethylammonium bromide (Sigma) and 1 mM phenylmethylsulfonyl fluoride . Homogenization of the hyphae was performed with a mini beadbeater (Biospec Products, Bartlesville, Okla.) in the presence of 0.5 g of glass beads by using three 20-s bursts . The homogenate was centrifuged at 10,000 x g for 15 min, and the supernatant was dialyzed as described above and then freeze-dried and resuspended in 50 µl of sterile distilled water .

Samples (2.5 µg for secreted proteins and 100 µg for nonsecreted proteins) were loaded onto a sodium dodecyl sulfate-12.5% polyacrylamide gel (Mini-Protean II; Bio-Rad) and electrophoresed for 1.5 h at a constant voltage (100 V) . Western blot analysis was performed with PL antiserum as previously described (26, 30) . To determine the total protein pattern (1), gels were silver stained (18) .

RNA extraction and Northern blot analysis.
Lyophilized hyphae were homogenized with a mini beadbeater (Biospec Products) in the presence of 1 g of Zirconia beads by using three 20-s bursts, and total RNA was extracted by using 1 ml of Tri-Reagent (Sigma) for every 25 mg of lyophilized hyphae . Following homogenization, samples were prepared according to the manufacturer's instructions (TRI-Reagent technical bulletin MB-205) . RNA was quantified by GeneQuant (Pharmacia Biotech, Cambridge, United Kingdom) .

Northern blot analysis was conducted by running 10 µg of total RNA on a 1.1% formaldehyde denaturing agarose gel (18) . The RNA was blotted onto a Hybond+ nylon membrane (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) by the capillary method (18) with 20x SSC (1x SSC is 17 mM NaCl plus 170 mM sodium citrate) . The RNA was fixed by baking the preparations for 2 h at 80°C and then subjected to hybridization . All hybridizations were carried out at 65°C, and all preparations were washed with 0.1x SSC . Probes were synthesized by using a Prime-a-Gene labeling system (Promega, Madison, Wis.) with [32P]dCTP . The hybridization probes used were the 1.1-kb pelB full-length clone (GenBank accession no . U32942), the pac1 coding sequence from C . gloeosporioides, and the ribosomal DNA (rDNA) repeat sequence from Neurospora crassa from pMF2 (9) .

The washed blot was exposed to a Fuji Bio Analyzing System sample screen . Images were captured with a Fuji Bio Analyzing System reader (Fujifilm, Tokyo, Japan) . Hybridization signals were quantified with MacBAS software, version 2.3 (Fujifilm) . The relative level of signals in each lane was corrected based on the background intensity and hybridization signal from the rDNA probe .

Nucleotide sequence accession number.
A partial sequence of the pac1 homologue of C . gloeosporioides has been deposited in a database under accession no . AF539700 .


   RESULTS

 
Effect of pH on the extracellular protein secreted by C . gloeosporioides.
Growth of C . gloeosporioides in M3S medium induced neither secretion nor expression of PL . Mycelia of C . gloeosporioides transferred from such primary cultures at an average pH of 5.0 to fresh glucose-buffered secondary medium with a pH of 4.0 resulted in a time-dependent increase in pH from 4.0 to about 6.0 within 10 h . Mycelia transferred to glucose-buffered secondary medium at either pH 4.0 or pH 6.0 did not cause any significant change in the medium pH . The amounts (Table 1) and patterns (Fig . 1) of extracellular proteins secreted in the two buffered media were significantly different . At all time points, 25 to 60% more total protein was detected in the culture medium at pH 6.0 than in the culture medium at pH 4.0 (Table 1), and additional protein species were synthesized at pH 6.0 that were absent at the lower pH (Fig . 1) .


TABLE 1 . Protein secretion during growth of C . gloeosporioides in buffered medium

 

FIG . 1 . Protein secretion during growth of C . gloeosporioides in inducing buffered media . The culture medium was concentrated, dialyzed, and analyzed as described by Bradford (1) . The pattern of secreted proteins observed after 12 h of incubation in buffered medium was analyzed by sodium dodecyl sulfate—12.5% polyacrylamide gel electrophoresis and silver staining . The numbers on the left are the molecular masses of the protein size markers.

 
Relationship among extracellular pH, pelB transcript levels, and PL secretion.
C . gloeosporioides mycelium transferred to unbuffered secondary medium at pHs ranging from 4.2 to 6.3 expressed detectable pelB beginning at pH 4.9 . The level of expression increased up to pH 6.0, but no further increase in transcript levels occurred at pH values above 6.3 (Fig . 2A, pelB panel) . PL secreted into the medium was first detected at pH 5.4, and maximal secretion was observed at pH 6.0 (Fig . 2A, PL panel) .


FIG . 2 . Transcriptional activation of pelB and PL secretion in buffered and nonbuffered secondary media . The growth medium for C . gloeosporioides was either not buffered (A) or buffered to pH 6.0 (B) . The culture media were harvested after 18 h . Hyphae were subjected to RNA extraction, and the culture medium was concentrated and dialyzed before analysis . Hybridizations were carried out at 65°C; blots were probed with the full-length cDNA pelB clone, stripped, and reprobed with the rDNA probe . PL from the culture medium was analyzed by Western blotting.

 
At a buffered constant pH of 6.0, pelB transcripts were detected 8 to 10 h after transfer to the medium, and the amount increased up to 8 h later (Fig . 2B, pelB panel) . PL secretion was observed concomitant with transcript accumulation, and maximum expression occurred 18 h after initiation of induction (Fig . 2B, PL panel) . At a constant pH of 4.0, neither secretion of PL nor expression of pelB transcripts was detected, even after 20 h of incubation (results not shown) .

pH regulation of pelB and pac1.
Five repeats of the sequence GCCAAG, the core binding sites for PacC (22), were found in the pelB promoter sequence . These repeats are situated at positions -547, -493, -410, -368, and -262, relative to the translation initiation codon (ATG) . Another four repeats in the antisense orientation (CGGTTC) are situated at positions -275, -282, -427, and -570 .

The influence of the ambient pH on the accumulation of transcripts encoding pelB and pac1 was examined by Northern blot analysis with a 254-bp pac1 probe located 288 bp upstream of the initiation codon . Mycelia were transferred from primary cultures with an average pH of 5.0 into fresh buffered secondary media with pHs ranging from 4.2 to 6.3, and this was followed by Northern blot analysis with a pac1 probe (Fig . 3A, pac1 panel) . Sixteen hours after induction, pac1 accumulation was greatest at pH 6.0 to 6.3 and was barely detectable at pH 4.2 (Fig . 3A) . When the C . gloeosporioides mycelium was transferred from primary medium to fresh secondary buffered medium at pH 4.0, neither pac1 nor pelB transcripts nor PL secretion was detected (Fig . 3B) . However, if the hyphae were transferred to secondary medium buffered at pH 6.0, initial pac1 transcripts were detected 8 h after transfer, while pelB transcripts and PL secretion were detected only after 20 h of incubation (Fig . 3B) . PL was detected inside the hyphae only after 20 h of induction in cultures grown under secreting conditions (data not shown), whereas there was no accumulation of PL in hyphae under nonsecreting conditions (results not shown) .


FIG . 3 . Transcriptional activation of pelB and pac1 by C . gloeosporioides as a function of pH levels . (A) Expression as a function of pH: Northern analysis of total RNA isolated from C . gloeosporioides mycelia 16 h after transfer to fresh secondary cultures buffered with phthalate . Blots were probed with pelB (pelB panel) and then sequentially stripped and reprobed with pac1 (pac1 panel) and rDNA probes . (B) Expression as a function of time and pH . Growth medium was buffered with phthalate to pH 4.0 or 6.0 or was not buffered . Cultures were harvested, hyphae were subjected to RNA extraction, and Northern blots were probed with pelB and then sequentially stripped and reprobed with pac1 and rDNA probes . A Western analysis of secreted PL was performed by using concentrated, dialyzed culture medium.

 
Effect of nitrogen on PL secretion.
Mycelia from C . gloeosporioides transferred from primary medium to buffered secondary medium at different pHs in which glutamine was the exclusive nitrogen source enhanced PL secretion as the pH increased to values above 5.4 (Fig . 4A) . Similar induction of secretion was obtained when mycelia were transferred to buffered secondary medium at pH 6.0 containing organic nitrogen sources, such as glutamate and glutamine, or inorganic nitrogen sources, such as NH4Cl . Similar results were obtained with NH4H2PO4 and Mg(NO3)2 (results not shown) . In contrast, no PL secretion was observed if the fungus was transferred to secondary medium at pH 4.0 (Fig . 4B) or to medium lacking a nitrogen source at pH 6.0 (results not shown) . An increase in the glutamate concentration from 0, 6.3, 12.5, or 25 mM in a buffered secondary medium at pH 6.0 was accompanied by an increase in the secretion of PL (Fig . 4C) .


FIG . 4 . Effect of the nitrogen source on PL secretion by C . gloeosporioides . (A) PL secretion as function of pH in the presence of 25 mM glutamine as the exclusive nitrogen source . (B) PL secretion as a function of pH 4.0 and 6.0 and nitrogen sources . Secondary medium was buffered with phthalate to different pHs in the presence of nitrogen sources, as indicated . Sixteen hours after transfer to the medium indicated, secreted PL was analyzed by Western analysis . (C) Effect of glutamate concentration on PL secretion . Mycelia were transferred into secondary medium at pH 6 with glutamate as the nitrogen source . After 20 h Western analysis was performed as described above.

 

   DISCUSSION

 
C . gloeosporioides is a broad-host-range phytopathogenic fungus that produces high levels of ammonia as a mechanism of tissue alkalinization . This environmental response was found in a large number of Colletotrichum species and isolates of the same species (14; Prusky, unpublished data) . Tissue alkalinization activates gene expression and regulates protein secretion (14) . A high pH (pH 6.0) significantly enhances both the quantity and the activity of the specific protein species secreted compared to the quantity and the activity of the proteins secreted at pH 4.0 . The pattern of pelB expression under several buffered conditions confirmed the previous findings of Yakoby et al . (30), who showed that alkalinization enhanced transcriptional activation of pelB . The alkaline environment also enhances the secretion of PL by C . gloeosporioides that occurs at higher pHs than the pHs that induce pelB . These results suggest that pH effects on pelB transcription determine PL secretion .

Transcriptional regulation of host-degrading enzymes by ambient pH has been demonstrated in the entomopathogenic fungus Metarhizium anisopliae (20) . In M . anisopliae, the kinetics of extracellular protease and chitinase transcript accumulation is pH dependent, and the expression patterns parallel the pH optima of enzyme activities . Similarly, in S . sclerotiorum, there is pH-based regulation with respect to oxalic acid production and pg1 expression (17) . Thus, a dynamic system of gene regulation based on ambient pH sensing and modification of the ambient pH environment may play a critical role in determining the pathogenic success of fungal pathogens such as C . gloeosporioides, M . anisopliae, S . sclerotiorum, Alternaria alternata (6), and possibly other phytopathogenic fungi .

The finding that pH plays a major role in regulation of pelB expression suggests that there is an ambient pH signal transduction pathway in C . gloeosporioides . Such a pathway has been characterized in A . nidulans and S . sclerotiorum, and several components of this pathway, including the gene for the pH-dependent transcriptional regulator PacC, have been cloned and characterized (24) . PacC homologs have also been identified in closely related filamentous fungi (12) and in yeast (10, 15, 21, 28) . Conservation of the zinc finger region and the central role that PacC plays in mediating pH-dependent signaling make a pacC homolog in C . gloeosporioides the prime candidate for regulating this process . Expression of pac1 in C . gloeosporioides is regulated by pH, and there is a steady increase in the transcript level, which parallels the increase in pelB expression, as the pH of the medium increases from 4.2 to 6.3 . Although sequence-specific DNA binding and regulation by proteolytic processing have not been demonstrated for pac1, the nine binding sites in the 5' upstream sequence of the pelB gene suggest that the pH-responsive pathway regulating the pacC gene in A . nidulans is present in C . gloeosporioides together with the other homologous components of this pathway .

The secretion of PL also is subject to nutritional signals, such as the presence of nitrogen . Regulation of nitrogen assimilation is complex and is important for disease development (13) . In C . gloeosporioides nutritional deprivation of primary nitrogen sources is critical for PL secretion . PL secretion was observed only when the pH was 6.0 or more and nitrogen, either inorganic [NH4Cl, NH4H2PO4, or Mg(NO3)2] or organic (glutamate or glutamine), was present . In vivo the nitrogen source for the pathogen's initial growth probably results from the activity of fungal proteases that break down the structural glycoprotein of the plant cell wall (16) . C . gloeosporioides, C . acutatum, and C . coccodes probably all utilize this source to secrete significant amounts of ammonia both under in vitro conditions and in decayed tissue (14) . Ammonia may have two key functions: (i) alkalization of tissue and (ii) direct or indirect (as a result of its transformation to glutamate and glutamine [13, 23]) activation of PL secretion (14, 30) . The mechanism by which the nitrogen signal activates PL expression and secretion could result from a specific type of regulation or from a general stress effect . Since increasing the amount of glutamine added to the growth medium results in increased PL secretion, we think that the general stress effect is the more likely explanation .

Our results demonstrate that the ambient pH is a major regulator of C . gloeosporioides pelB transcriptional activation and PL secretion . PL secretion is transcriptionally regulated by pH, and no significant accumulation of PL protein was detected in the hyphae . At least one component of a conserved regulatory pathway mediating pH-regulated gene expression, pac1, exists in this fungus . Our data suggest that alkalization and the presence of nitrogen are important factors for C . gloeosporioides attack and development in a fruit host . Targeting ammonia accumulation, which results in alkalinization, and other pH-regulated processes, such as pelB expression, may result in a viable strategy for blocking development of disease caused by this plant pathogen .

 


   ACKNOWLEDGMENTS

 
N . Drori and H . Kramer-Haimovich contributed equally to this work .

This research was supported by research grants to D.P . from BARD and the Israel Science Foundation . N.D . and H.K.-H . were awarded personal scholarships from the Israel Fruit Marketing Board/Ministry of Agriculture & Rural Development .

We thank D . Beno-Moalem for advice during early periods of this work .


   FOOTNOTES

 
* Corresponding author . Mailing address: Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel . Phone: 972 3 9683610 . Fax: 972 3 9683622 . E-mail: dovprusk{at}agri.gov.il .


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