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Differential expression of mepA, mepC and smtE during growth and development of Microbotryum violaceum

     Department of Biology, University of Louisville, Louisville, Kentucky 40292

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Many fungi require a dimorphic switch from budding to filamentous growth to cause infection. Although the control of dimorphism has been elucidated for organisms such as Saccharomyces cerevisiae and Ustilago maydis, almost nothing is known about the control of mating and dimorphism in Microbotryum violaceum. M. violaceum mepA, mepC and smtE are homologs of genes whose encoded products act as, or interact with, components of the MAPK and cAMP-PKA pathways, conserved pathways that regulate mating and dimorphism in other fungi. A comparison of gene expression under various in vitro conditions was superimposed on a comparison of in vitro vs. in planta expression to yield a more complete picture of the expression of these genes in M. violaceum during fungal development. For the most part the expression of these genes was highest on low ammonium, intermediate for mated and in planta, and lowest on rich medium. As expected, under conditions of low ammonium, expression of the M. violaceum ammonium permease genes mepA and mepC mirrors that of S. cerevisiae MEP2 and U. maydis ump2. An intriguing possibility is that MepA is a sensor to signal when conditions are conducive for mating. The upregulation of smtE, which encodes a PAK kinase, suggests that the MAPK pathway regulates, at least partially, mating and might be linked to ammonium sensing/transport in M. violaceum.

Key words: ammonium transport, fungal dimorphism, plant pathogenesis

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Microbotryum violaceum Deml and Ober winkler (=Ustilago violacea [Pers.] Fuckel) is a heterobasidiomycete plant pathogen with some interesting features. Although more than 200 Caryophyllaceae serve as hosts, individual M. violaceum isolates are restricted in their host range to one or a few species (Thrall et al 1993). Dramatic divergences among isolates detected by a phylogenetic analyses of a variety of molecular genetic characters recently has prompted several authors to suggest that M. violaceum is actually a species complex, rather than a single fungal species (Freeman et al 2002, Perlin et al 1997, Bucheli et al 2000, Hood et al 2003).

M. violaceum must complete its life cycle in a host plant. The dark teliospore masses give the flowers a smutted appearance, thus earning M. violaceum the name "anther smut." Infective teliospores are dispersed by air or by insects (Alexander 1989) where they may land on the moist surface of a compatible host plant. Here they undergo meiosis yielding haploid sporidia of opposite mating types (a1 and a2). The sporidia also can be propagated in the laboratory on artificial media as budding cells. The initial union of the two haploid cells, followed by perception of appropriate host signals, results in a dimorphic switch producing infective dikaryotic hyphae that penetrate the surface of compatible hosts. Once inside the hyphae grow principally through the plant stems until they reach the bud meristems and anthers (Akhter and Antonovics 1999). Here the nuclei fuse (karyogamy) and teliosporogenesis occurs, thus completing the life cycle (Kokontis and Ruddat 1986, Ruddat et al 1991, Scutt et al 1997).

The control of mating and filamentous growth via the mitogen-activated protein kinase (MAPK) and cAMP-dependent protein kinase A (PKA) pathways has been elucidated for S. cerevisiae but the signals, mechanisms and outcomes of this control have been shown to differ among other fungi, including Schizosaccharomyces pombe and Ustilago maydis (Lengeler et al 2000, Gold et al 1994). The only published data for the molecular mechanisms involved in the dimorphic switch for M. violaceum are for the novel genes HSGc11 (Wang and Perlin 1998) and MIGc34 (Perlin and Wang 1997). No data have been published for gene expression during infection. Moreover, because there is a paucity of genomic sequence data for this organism, techniques such as micro-array analysis are unavailable. The M. violaceum genes mepA and mepC encode putative ammonium transporters/sensors whose homologs in S. cerevisiae play important roles in signal transduction pathways controlling filamentous growth. Similarly smtE encodes a M. violaceum homolog of STE20, an upstream component of both the mating and filamentation pathways in S. cerevisiae.

The goal of this study was to compare the expression of mepA, mepC and smtE during discrete stages of fungal development and thereby gain insights into the control of such developmental programs in M. violaceum.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Fungal strains, growth media and plant infection.— – The M. violaceum sporidial stains A1.28, mating type a1 and A2.28, mating type a2 were provided by M.E. Hood, Duke University, North Carolina. The laboratory media used were Y PD (yeast peptone dextrose, Castle and Day 1984), SLAD (synthetic low ammonium dextrose, Gimeno et al 1992), and water agar (2% agar). Infection of Silene latifolia strain "S1," provided by M.E. Hood, University of Virginia, followed established protocols (Akhter and Antonovics 1999).

M. violaceum and S. latifolia genes and PCR.— – All M. violaceum genes used as probes and for analyses of gene expression were isolated and characterized either as described by our laboratory (ß-tub, Shi and Perlin 2001; mepA and mepC, Smith et al 2003) or by PCR with degenerate primers (smtE). Sequence comparison of a 1471 bp fragment for both cDNA and genomic DNA indicated that this portion of the smtE gene contains no introns (W. Hong, masters thesis, University of Louisville, 2001). Southern analysis indicates that ß-tub, mepA, mepC and smtE are single copy genes. As mentioned earlier, mepA and mepC encode putative ammonium permeases, while smtE is predicted to encode a homolog of the PAK kinase, Ste20p. The ß-tub gene encodes ß-tubulin, a highly conserved component of the mitotic spindle apparatus. As such it is considered a housekeeping gene, suitable for use as a loading standard for Northern analyses of phytopathogen gene expression (Montanini et al 2002, van der Vlugt-Bergmans et al 1997). In addition ß-tub amplification was used to standardize the semiquantitative RT-PCR results. Initial comparisons with another housekeeping gene, that encoding TFIID, provided similar results, confirming that ß-tub was a valid gene to use for standardization.

PCR primers specific to M. violaceum genes ß-tub, mepA, mepC and smtE were used to generate Northern hybridization probes and to compare gene expression via RT-PCR. The sequences for primers used and the respective conditions for amplification are available on request. The sequence identity for each PCR product to be used as a probe was confirmed by direct DNA sequencing of PCR products purified from agarose or after cloning into pCR2.1 TOPO vector (Invitrogen, Carlsbad, California). DNA sequencing used the BigDye direct sequencing kit (ABI/Perkin Elmer, Foster City, California), and products were analyzed on a 310 Genetic Analyzer (ABI).

Manipulations involving RNA from plants and fungal cells.— – The TRIzol (Invitrogen) method for total RNA isolation was used in all cases. The four fungal in vitro samples used for Northern analysis were "rich" (from Y PD, rich medium), "mated" (a mixture of approximately 40% mated + approximately 60% nonmated cells resulting from mating induction), "nonmated" and "low NH₄⁺" (from SLAD medium). Before RNA isolation strains/samples were grown and prepared as follows. "Rich": strains A1.28 and A2.29 were grown on Y PD 5 d at room temperature, 3 g of each were used for separate RNA isolations. "Mated": strains A1.28 and A2.29 were grown 5 d on Y PD, induced to mate by incubation on water agar 2 d at 15 C. The presence of mated cells was verified microscopically, and 3 g of the mated cell mixture was used for RNA isolation. "Nonmated": s trains A1.28 and A2.29 were grown 5 d on Y PD then incubated separately on water agar 2 d at 15 C. Approximately 1.5 g of each of the two SLAD-grown strains was combined for RNA isolation. "Low NH₄⁺": strains A1.28 and A2.29 were grown 6 d at room temperature on SLAD. Then approximately 1.5 g of each of the two SLAD-grown strains was combined for RNA isolation. For both Northern analyses and RT-PCR, at least two independently isolated RNA samples were used for each determination.

The RNA samples were fractionated in a formaldehyde gel, electrophoresed and transferred to Nytran nylon membranes (Schleicher & Schuell, Keene, New Hampshire) following the method described by Sambrook et al (1989) with modifications according to the manufacturer. The membranes were UV cross-linked at 150 mJ and baked 2 h at 75 C. All hybridization steps were performed according to the manufacturer’s protocol for the ULTRAhyb Hybridization Buffer method (Ambion, Austin, Texas). The Personal Molecular Imager (BioRad, Hercules, California) was used for detection; the Quantity One Quantitation Software (Bio-Rad) and Microsoft Windows Excel program were used for data analyses. The M. violaceum ß-tub gene was used as a loading standard and for normalization of results (Montanini et al 2002).

Semiquantitative RT-PCR.— – Two µg of total RNA from mated fungal, infected plant (mixed bud) and noninfected plant were used as starting template for cDNA synthesis with the RETROscript First Strand Synthesis Kit for RT-PCR (Ambion). Appropriate control PCR reactions were used to demonstrate lack of contamination with genomic DNA, as well as lack of amplification from noninfected plant cDNA of fungal specific products. To draw conclusions from the measurement of expression in planta, a comparison sample was needed that would represent M. violaceum gene expression when it was not infecting the plant. By virtue of its life cycle all M. violaceum cells in planta are dikaryotic until teliospore formation, at which time they become diploid. Thus mated cells were used for RNA isolation to represent the in vitro fungal portion of the "in vitro" comparison sample. Also, because only a portion of the total RNA from infected plant tissue would be of fungal origin, this would necessitate diluting the cDNA produced from the 100% (mated) fungal RNA. To create additional similarity between the infected plant sample ("in planta") and the "in vitro" comparison sample, the dilution was made with non-infected plant cDNA. Thus, the "in vitro" cDNA template was a mixture of (noninfected plant cDNA + mated fungal cDNA). The "in planta" cDNA template was purified from mixed bud (sizes ranges of 1–22 mm) of infected plants. By dilution, the starting total cDNA concentrations of "in vitro" and "in planta" templates were matched so that they gave fungal ß-tub PCR amplification bands of equal intensities. These ß-tub-equalized "in vitro" and "in planta" samples then were used to compare PCR amplification band intensities for the genes of interest. The semiquantitative RT-PCRs were performed in duplicate, with two sets of ß-tub-equalized "in vitro" and "in planta" cDNA samples. These two cDNA sets were synthesized from two sets of total RNA samples (which were isolated from duplicate plant tissue aliquots and fungal cultures). The PCR amplification of both ß-tub and of the specific genes of interest was followed at increasing numbers of cycles of amplification by electrophoresis in 1% agarose/1x TAE that contained ethidium bromide stain (Sambrook et al 1989). In this way we were able to determine that amplification had not yet reached plateau and compare signal intensity for the in vitro vs. in planta samples.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Mating and the dimorphic switch are linked to fungal pathogenesis. To study gene expression associated with this switch total RNA for M. violaceum was obtained under various in vitro conditions and in planta. In vitro conditions included growth on rich medium (Y PD) and on low ammonium (SLAD). Comparison also was made for RNA from mated vs. non-mated cells. In planta RNAs were isolated from infected or noninfected bud tissue.

Northern analyses of gene expression.— – A series of Northern analyses was begun to measure the expression of M. violaceum mepA, mepC and smtE. The initial Northern series included in planta RNA samples, but several different Northern protocols failed to provide sufficient sensitivity to analyze the expression from in planta samples (data not shown). This could be attributed to low percent fungal RNA in planta, low gene expression, interference with fungal hybridization signal by plant RNA or by other inhibitors present in the plant RNA sample.

Northern analyses did provide sufficient sensitivity to analyze fungal gene expression outside the plant. Such analyses revealed increased expression of mepA on low ammonium as compared to rich medium and in comparison with nonmated cells (TABLE I). Although mepA was up-regulated for mated vs. non-mated samples, the greatest upregulation was seen for low ammonium (on average, 51-fold higher for low ammonium as compared to rich medium).

Northern analyses of smtE expression were performed in duplicate and indicated an approximately fourfold upregulation for the low ammonium sample vs. rich sample and slight upregulation (1.5-fold) for mated vs. nonmated samples (TABLE I). The small difference in expression for mated vs. nonmated was mirrored in semiquantitative RT-PCR results.

Semiquantitative RT-PCR.— – Data have been published in which fungal pathogen gene expression was measured in vitro and/or in planta by comparing the agarose gel band intensities of PCR products amplified from cDNA templates (Carlile et al 2000, Eddine et al 2001, Tenberge et al 1996).

Comparison of mepA expression for in vitro vs. in planta samples revealed no marked differences (FIG. 1). There appeared to be a trend for slight upregulation of mepC in planta; and there were no marked differences seen for the expression of smtE for in vitro vs. in planta samples. However the slight difference in expression observed in Northern analyses for mated vs. nonmated cells also was observed. For smtE, the RT-PCR band intensity ratio observed in agarose gels for mated vs. nonmated samples respectively was 1.2 vs. 1 (at 23 cycles) and 1.4 vs. 1 (at 24 cycles).

View larger version (46K): [in this window] [in a new window]   FIG. 1. A comparison of in planta vs. in vitro expression for mepA, mepC and smtE A–D. In planta (IP) and in vitro (V) cDNA template pairs were amplified respectively with ß-tub, mepA, mepC and smtE primers. The equalized ß-tub signals for IP vs. V indicate equal amounts of starting cDNA template for these samples. A number over a bar indicates the number of cycles of amplification for paired samples. Noninfected plant cDNA template (N) was included as a negative control and in each instance failed to yield the target amplicon. The unlabeled lane was loaded with Hi-Lo DNA marker (Minnesota Molecular, Minneapolis, Minnesota). The comparisons of expression for ß-tub, mepA, mepC and smtE were based on signals obtained after 25, 33 and 28 cycles of amplification, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

MepC expression was highest for low ammonium, intermediate for in planta and mated (although in planta expression was slightly higher than that for mated). The mepC expression for mated was observed to be slightly higher, or equal to, the expression for rich and nonmated. smtE expression was highest for cells grown on low ammonium, intermediate for mated and in planta and lowest for non-mated and rich conditions.

The dimorphic switch in M. violaceum requires mating and the transition from budding to filamentous growth. However no results have been published for the molecular mechanisms governing mating or the dimorphic switch in M. violaceum other than our previous reports of two genes whose expression was increased during mating relative to nonmated cells (Perlin and Wang 1997, Wang and Perlin 1998). Lorenz and Heitman (1998) proposed a model for the regulation of pseudohyphal growth of S. cerevisiae via an ammonium transporter, Mep2p, and the PKA pathway.

Smith et al (2003) reported the isolation and characterization of four MEPs from smut fungi: M. violaceum mepA and mepC, and U. maydis ump1 and ump2. Consistent with their amino acid similarity to Mep2p, MepAp and Ump2 were determined to be high affinity transporters. Of the three genes characterized fully, only ump2, but not ump1 or mepA, could restore pseudohyphal growth in yeast triple MEP mutants (i.e. those lacking all three genes encoding this family in S. cerevisiae). Based on these similarities between Ump2p and Mep2p one might guess that expression of ump2 would be up-regulated on low ammonium. In fact expression of ump2 was elevated at concentrations of ammonium between 0.5 and 2 mM and reduced at concentrations of ammonium greater than 2 mM (Smith et al 2003). Also, like S. cerevisiae, U. maydis has a budding phenotype on rich medium and a filamentous phenotype on low ammonium (Smith et al 2003). Upregulation of M. violaceum mepA and mepC on low ammonium is consistent with that of ammonium transporters in S. cerevisiae (MEP2) and U. maydis (ump2), but the absence of filamentous phenotype indicates that M. violaceum requires another cue, in addition to low ammonium, to affect a dimorphic switch.

The upregulation of mepA expression for mated vs. nonmated cells is intriguing. No data have been published on upregulation of fungal ammonium permeases/transporters during mating. Perhaps the upregulation of mepA expression during mating represents a cue necessary for mating. For S. cerevisiae and U. maydis, mating occurs between haploids of opposite mating type on rich medium, although nitrogen limitation might play a role for U. maydis (Lengeler et al 2000). Thus, although a pheromone trigger is required, mating does not absolutely require a nutritional trigger (e.g. low ammonium and/or low fermentable carbon source). In contrast, in the fission yeast S. pombe both nutrient starvation (especially nitrogen) and availability of mating pheromone are required for mating (Leupold 1987). In vitro mating between M. violaceum a1 and a2 haploids similarly can take place only on starvation medium at 15 C, an indication that a combination of pheromone and nutritional cues is required for mating. Perhaps mepA plays a pivotal role in linking the nutritional cue of low ammonium to the regulation of mating in M. violaceum.

Northern analyses suggest that the upregulation of mepA on low ammonium might be significantly higher than that for mepC. This observation, together with the finding that mepA but not mepC is up-regulated for mating cells, suggests distinct functions for these ammonium transporters. There appeared to be a trend for slight upregulation of mepC in planta. If true, then the role of mepC as an ammonium transporter in planta also might be distinct from that of mepA. Published data support a model for control of nitrogen metabolism via sets of ammonium transporters/permeases that have nonredundant functions (Marini et al 1997, Javelle et al 2001, Monahan et al 2002).

M. violaceum smtE is a homolog of S. cerevisiae STE20 and S. pombe SHK1 (Tu and Wigler 1999). Mutation of STE20 results in failure of pseudohyphal differentiation (Liu et al 1993) and sterility in S. cerevisiae, while in S. pombe loss of Shk1 is lethal (Yang et al 1999). Disruption of the U. maydis STE20 homolog, smu1, severely attenuates both mating and pathogenicity (Smith et al 2004). If smtE indeed is up-regulated for mating cells, mating pheromones might play a direct or indirect role in the positive regulation of smtE expression. The nutritional cue of low ammonium similarly might play a role in increased expression of smtE. If the role of M. violaceum SmtE is analogous to that of S. cerevisiae Ste20p, then increased SmtE might be required to affect an increase in mating and/or filamentous growth. No marked differences were seen for the expression of smtE for in vitro vs. in planta samples. This might reflect the fact that M. violaceum cells already are growing filamentously in infected bud tissue (Uchida et al 2003). If its role were to stimulate dikaryon formation, smtE would not be expected to be further up-regulated in planta.

While M. violaceum has served as a model organism in the study of host-pathogen dynamics (Alexander et al 1996, Bucheli et al 2000) and transmission genetics (Garber and Ruddat 2002), it also has been the subject of molecular genetic studies aimed at characterization of gene expression and function. The decision to include this fungus on the list of organisms for genome sequencing by the Broad Institute is a reflection of the importance of M. violaceum as a research organism. Many fungal pathogens of plants and humans are dimorphic and require a switch from a yeast-like state to a hyphal state to cause disease. The design of effective strategies against these pathogens thus requires an understanding of such transitions at the molecular level. This investigation has shed light on some of the similarities and differences between M. violaceum and other fungi in the expression of genes whose encoded products act as, or interact with, components of conserved pathways that regulate fungal mating and dimorphism.

	ACKNOWLEDGMENTS

 
This article is dedicated to the honor and memory of David G. Smith. David was always incredibly generous with his time. His willingness to share his infectious intellectual curiosity and laboratory skills helped to mold many a student, colleague (and major professor!). The loss at an early age of such a promising scientist and friend is one that will be felt by us all. The authors also wish to thank Michael E. Hood and Shamima Akhter for providing the S. latifolia S1 seeds and the M. violaceum haploid strains A1.28 and A2.29. We thank Janis Antonovics and Peter H. Thrall for their guidance on the inoculation and cultivation techniques that were used for plant infection. This work was supported in part by an IRIG-RIG to M.H.P. from the Office of the Vice President for Research at the University of Louisville and by a grant to C.F.H. from Sigma Xi.

	FOOTNOTES

 
Accepted for publication January 3, 2005.

¹ Corresponding author. E-mail: mhperl01{at}gwise.louisville.edu

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