Role of LAMMER Kinase in Cell Wall Biogenesis during Vegetative Growth of Aspergillus nidulans.

Depending on the acquisition of developmental competence, the expression of genes for β-1,3-glucan synthase and chitin synthase was affected in different ways by Aspergillus nidulans LAMMER kinase. LAMMER kinase deletion, ΔlkhA, led to decrease in β-1,3-glucan, but increase in chitin content. The ΔlkhA strain was also resistant to nikkomycin Z.

Fungal cells constantly remodel their rigid structure by reflecting differences in types of cell wall-related genes (CWGs) and their transcriptional regulation through cell signaling in respond to external stimuli and the cell cycle [1, 2]. In fungi, the cell wall integrity signaling (CWIS) pathway is responsible for cell wall remodeling and reinforcement following cell wall stress. The CWIS pathway in Saccharomyces cerevisiae is sequentially stimulated through the stress sensors Wsc1p and Mid2p; the GTP-binding protein, Rho1p; protein kinase C; the mitogen-activated protein kinase, mitogen-activated protein kinase (MAPK), cascade; and two types of transcription factors, e.g. MADS-box transcription factor Rlm1 and SBF (cycle box-binding factor) proteins Swi40 and Swip6 [3]. Although several homologous CWIS elements have been identified in Aspergillus nidulans [4], studies of cell wall biogenesis and regulation of the transcription of cell wall-related genes have been limited [2, 5]. Unlike in S. cerevisiae, the transcription of most CWGs in A. nidulans seems to be regulated by non-MpkA, a MAPK, signaling, with the exception of the α-1,3-glucan synthase genes (agsA and agsB) [2].

The dual-specificity LAMMER kinases are involved in many cellular processes, including differentiation, the cell cycle, stress response, and reproduction. In A. nidulans, one of the prominent features revealed by deletion of the LAMMER kinase gene (lkhA) was the morphological changes during vegetative growth and various developmental stages. The patterns of germ tube emergence and hyphal polarity were altered and septation was increased by the deficiency of LAMMER kinase, which led to a detrimental effect on asexual and sexual differentiation [6]. In fission yeast, LAMMER kinase, Lkh1, regulates G1/S progression of the cell cycle by modulating the activity of cyclin-dependent kinase inhibitor, Rum1 [7]. Our previous reports also revealed that expression of the genes for the synthesis of chitin and β-1,3-glucan is cell cycle-dependent and developmental stage-specific [8, 9, 10, 11, 12]. From these results, it was postulated that LAMMER kinase may be involved in the non-MpkA signaling of CWGs in A. nidulans. In order to test this possibility, we analyzed the expression of CWGs, the composition of the major cell wall polysaccharides chitin and β-1,3-glucan, and the susceptibility to cell wall-damaging agents using a LAMMER kinase deficient strain (ΔlkhA) of A. nidulans [6].

A. nidulans strain DLA1 (yA2; argB2; pyroA4; veA+; ΔlkhA::argB), the LAMMER kinase deletion strain [6], and MCBA001 (yA2; pyroA4; veA+), the meiotic progeny produced by the cross between A4 and TJ1 (yA2; argB2; pyroA4; veA+), were used in this study. A. nidulans complete medium was prepared and used for culture and preparation of vegetative mycelia by the previously described method [6].

RNA preparation was performed as described previously [6]. Briefly, the cultured cells were harvested and ground to a powder with a mortar and pestle in liquid nitrogen. GT buffer (4 M guanidine thiocyanate, 25 mM sodium acetate [pH 6.0], 0.5% N-lauroylsarkosine, 0.84% β-mercaptoethanol) was added to the cell powder. After vortexing, the debris was removed by centrifugation. The supernatant was then overlaid with 2mL of 5.7M CsCl solution (5.7M CsCl, 25 mM sodium acetate, pH 6.0). The RNA pellet was isolated by centrifugation at 10℃, 125,000 ×g for 16 hr using an SW 55Ti rotor (Beckman Coulter, Brea, CA, USA). The pellet was resuspended in 400 µL TES buffer (10 mM Tris-Cl, pH 7.4, 5 mM EDTA, 1% sodium dodecyl sulfate) and extracted with phenol/chloroform (pH 4.5). The RNA was concentrated by ethanol precipitation, dissolved in DEPC-treated water, and stored at -70℃.

Relative mRNA levels were determined by quantitative real-time (qRT) PCR using the specific primer sets designed for each gene (Table 1). Briefly, total RNA was reverse transcribed into cDNA using the M-MLV Reverse Transcriptase (Elpis Biotech, Inc., Daejeon, Korea) according to the manufacturer's instructions. Each gene-specific primer was optimized for expression analysis through RTPCR on a CFX96 Real-Time PCR (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using TOPreal qPCR 2× PreMIX (Enzynomics, Daejeon, Korea). The PCR cycling parameters were 5min at 95℃, followed by 45 cycles of 20 sec at 95℃, 20 sec at 57℃, and 15 sec at 72℃. A melting curve analysis to confirm specificity was performed for each primer set. Gamma-actin (gAct) was used for normalization, and the relative expression was calculated according to the method previously reported [13].

Table 1

Oligonucleotides used for qRT-PCR analysis

qRT-PCR, quantitative real-time polymerase chain reaction.

Chemical analysis of the composition of the cell wall was performed using the method described previously [14]. Sensitivity toward cell wall-perturbing agents was investigated via the method described by Kovács et al. [5] using media supplemented with Calcofluor white (CFW) or Congo red (CR). Disk diffusion method [15] was used to evaluate the effect of antifungal drugs, nikkomycin Z and terbinafine.

A previous study revealed that the genes for chitin synthase and β-1,3-glucan synthase are expressed in a cell cycle-dependent manner [8, 9, 10]. Similar to LAMMER kinase in fission yeast [7, 16, 17], the involvement of LAMMER kinase in cell division of A. nidulans was also indicated with fluorescent staining to visualize the septation and nuclear division in vegetative hyphae [6]. Therefore, we tested the effect of LAMMER kinase on the expression of genes for cell wall polysaccharides in vegetative hyphae via qRT-PCR. When the expression levels of the genes for chitin synthase (chsA, B, C, D, cmsA, and cmsB), α-1,3-glucan synthase (agsA and B), and β-1,3-glucan synthase (fksA) were quantified from the RNA of a 9-hr-old vegetative mycelia of DLA1, only fksA was discovered to be at a statistically significant diminished level (p = 0.012) (Fig. 1A). On the other hand, qRT-PCR of RNA from a 15-hrold vegetative mycelia of DLA1, which had already acquired developmental competence, the chsC and chsD genes for chitin synthase revealed statistically significant increase (p = 0.006) and decrease (p = 0.021), respectively (Fig. 1B). These results indicate that, in A. nidulans, LAMMER kinase positively regulates the expression of fksA before the acquisition of developmental competence, and LAMMER kinase affects the expression of only two chitin synthase genes, chsC and chsD, in different ways after the acquisition of developmental competence during vegetative growth; chsC was negatively but chsD was positively regulated. It is also noteworthy that chsC and chsD have different functions; chsC is required for maintenance of hyphal wall integrity and development of asexual structures such as the conidiophores [18], whereas chsD plays a role in hyphal growth and development [19].

Fig. 1

Characterization of gene expression changes of cell wall-related genes. RNA was purified from vegetative cultures from 9-hr (A) and 15-hr (B) samples for quantitative real-time polymerase chain reaction (qRT-PCR). Three biological samples were taken for the first cDNA synthesis, and subsequent real-time PCR analysis was performed using specific primer sets for cell wall genes. Conditions for qRT-PCR are described in the text. The asterisk indicates statistically significant change due to the deletion of LAMMER kinase (ΔlkhA) as calculated with the Student's t-test for p ≤ 0.05.

Since changes in the expression of the genes for cell wall polysaccharide synthesis were apparent in the lkhA-deletion strain, the content of cell wall chitin and β-1,3-glucan were determined using a 15-hr-old mycelia from both the wildtype and DLA1. In good agreement with the results from Fig. 1, the amount of β-1,3-glucan was decreased by more than 20% in the DLA1 strain versus wild-type, but the amount of chitin was increased about 30% (Table 2). The increase of chitin contents in DLA1 can also be explained by a compensatory mechanism, which may be a back-up for the cell wall defects caused by a low level of fksA expression and, thus, of β-1,3-glucan content.

Table 2

Composition of cell wall polysaccharides

aChemical analyses of the cell wall preparations were performed three times with wild type (WT) and LAMMER kinase-deletion strain (ΔlkhA) cultured on complete solid medium at 37℃ for 15 hr. In each chemical analysis, samples for the carbohydrate assay were tripled.

In yeast, the binding of cell wall-perturbing agents to stress sensors activates signaling pathways like CWIS and high-osmolarity glycerol [20], and, therefore, modulates β-1,3-glucan [21] and chitin synthesis at a transcriptional level [22]. Sensitivity to cell wall-perturbing agents is indicative of the composition of the cell wall, depending on which component the agent reacts with. CFW interacts specifically with β-1,4-linkage of polysaccharides such as chitin. CR forms a complex with chitin and β-glucan and inhibits chitin synthases [5, 23]. Thus, we tested the sensitivity of our strains toward cell wall-perturbing agents such as CFW and CR. As shown in Fig. 2A, DLA1 revealed no significant difference in sensitivity to the β-glucan-specific CR as well as the chitin-specific CFW. Antifungal drug test, however, revealed that deletion of lkhA made the cells resistant to the nikkomycin Z, which is a specific inhibitor for type I chitin synthase such as ChsC [24], but not to the terbinafine, which inhibits ergosterol synthesis and thus affects cell wall synthesis (Fig. 2B).

Fig. 2

Sensitivity to cell wall-damaging agents. A, Identical volumes of 10-fold serial dilutions of exponentially growing wild-type cells and cells lacking LAMMER kinase (ΔlkhA) were spotted onto minimal medium (MM) containing 160 µg/mL of Congo red (CR) or Calcofluor white (CFW) and incubated at 37℃. Colony growth was inspected after 48-hr incubation at 37℃; B, The equal volume of conidia suspension (1 × 107 cells/mL) was spread uniformly onto MM agar plate. The 6 mmdiameter paper disk was saturated with 2 µg of terbinafine or nikkomycin Z and placed onto inoculated plate. Plates were incubated at 37℃ for 72 hr to observe the formation of inhibition zone.

In summary, the results presented here indicate that the A. nidulans LAMMER kinase, LkhA, affects the cell wall polysaccharide composition of vegetative hyphae by modulating the expression of the cell wall genes fksA, chsC and chsD. Although further studies are required, our results unlock interesting avenues for further investigation into the regulatory mechanism of the gene(s) for cell wall biogenesis in filamentous fungi.