AbSte7, a MAPKK Gene of Alternaria brassicicola, Is Involved in Conidiation, Salt/Oxidative Stress, and Pathogenicity

Abstract

Alternaria brassicicola (Schwein.) invades Brassicaceae and causes black spot disease, significantly lowering productivity. Mitogen-activated protein kinases (MAPKs) and their upstream kinases, including MAPK kinases (MAPKKs) and MAPKK kinases (MAPKKK), comprise one of the most important signaling pathways determining the pathogenicity of diverse plant pathogens. The AbSte7 gene in the genome of A. brassicicola was predicted to be a homolog of yeast Ste7, a MAPKK; therefore, the function was characterized by generating null mutant strains with a gene replacement method. AbSte7 replacement mutants (RMs) had a slower growth rate and altered colony morphology compared with the wild-type strain. Disruption of the AbSte7 gene resulted in defects in conidiation and melanin accumulation. AbSte7 was also involved in the resistance pathways in salt and oxidative stress, working to negatively regulate salt tolerance and positively regulate oxidative stress. Pathogenicity assays revealed that AbSte7 RMs could not infect intact cabbage leaves, but only formed very small lesions in wounded leaves, whereas typical lesions appeared on both intact and wounded leaves inoculated with the wild-type strain. As the first studied MAPKK in A. brassicicola, these data strongly suggest that the AbSte7 gene is an essential element for the growth, development, and pathogenicity of A. brassicicola.

Introduction

The phytopathogenic fungus Alternaria brassicicola (Schwein.) Wiltshire is the causal agent of black spot disease of Brassicaceae [2]. The disease is of worldwide economic importance, causing significant reductions in yield quantity and quality of the cultivated Brassica species [2]. For example, the incidence of black spot disease in Kenya on cabbage and kale farms was 30.3 and 8.7%, respectively, but A. brassicicola was the predominant pathogen [16]. As a typical necrotrophic fungal pathogen, the A. brassicicola-Brassicaceae and A. brassicicola-Arabidopsis thaliana pathosystem have been used as models for necrotrophic fungal-plant interactions [18]. Moreover, A. brassicicola is strongly associated with the development and onset of human chronic respiratory diseases [17].

Despite the great economic and scientific importance of this species, understanding of the pathogenesis mechanisms of A. brassicicola is limited. In past decades, mitogen-activated protein kinase (MAPK) signal transduction pathways have been reported in the pathogenesis of diverse plant pathogens [6,15,21,25]. Three MAPK genes associated with the MAPK pathways of A. brassicicola, Amk1, AbSlt2, and AbHog1, have been cloned and characterized. Disruption of Amk1, the Fus3/Kss1 homolog of yeast, resulted in complete loss of pathogenicity [4]. The AbSlt2 replacement mutants (RMs) and AbHog1 disruptant also had reduced virulence on host plants [8].

Although some progress has been made in elucidating the MAPK pathways in A. brassicicola, there are many unsolved problems regarding the upstream signaling pathways. To date, no studies have investigated MAPK kinases (MAPKKs) or MAPK kinase kinases (MAPKKKs) in A. brassicicola. The downstream transcription factors and target genes in these three signal transduction pathways are not known, except for the gene AbSte12 [5]. In this study, AbSte7 was identified as a putative MAPKK gene of A. brassicicola and a homolog of yeast Ste7 by genome BLAST searches comparing the yeast Ste7 protein sequence with the genome of A. brassicicola. A series of experiments were conducted to reveal the functions of the AbSte7 gene. To the best of our knowledge, this is the first report identifying the roles of an MAPKK gene in A. brassicicola relevant to development and pathogenicity.

 

Materials and Methods

Fungal Strains and Culture Conditions

A. brassicicola strain abta14 was isolated from infected leaves of cabbage (B. oleracea) in the Provence of Shandong, China and used as the wild-type (WT) strain in this study. For genomic DNA and RNA extraction, all strains were cultured on potato dextrose agar (PDA; Becton, Dickinson and Company, USA) at 25℃ for 5 days. For protoplast preparation, A. brassicicola was incubated in potato dextrose broth (Becton, Dickinson and Company) at 25℃ with shaking at 200 rpm for 36 to 48 h.

Identification of the AbSte7 Gene in A. brassicicola

The protein sequence of Saccharomyces cerevisiae Ste7 (NCBI Accession No. CAA98732) and its homolog, Pyrenophora tritici-repentis (NCBI Accession No. XP_001932876), were used to search for putative MAPKK genes in the A. brassicicola genome by a genome BLAST search (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi?organism=fungi). Gene-specific primers (Pf/Pr, Table 1) were designed based on the BLAST results and used to amplify the DNA and cDNA sequences of the AbSte7 gene from A. brassicicola WT abta14.

Table 1.Primers used in this study.

Targeted Gene Disruption and Functional Complementation of AbSte7

An AbSte7 replacement construct was generated with the double-joint PCR method described by Yu et al. [24]. Briefly, the truncated 5’ and 3’ ends of AbSte7 were amplified by PCR using the abta14 genomic DNA with two primer pairs, p1/p2 and p3/p4, respectively. Simultaneously, the selectable marker corresponding to the HygB gene cassette from the pUCATPH plasmid was amplified with the p5/p6 primer set. Nested PCR was conducted with the p7/p8 primers, and the resulting DNA fragments were purified and transformed into protoplasts prepared from the abta14 strain according to the protocol developed in our laboratory [20]. RMs of AbSte7 were selected on PDA with 50 μg/ml hygromycin (Invitrogen, USA).

AbSte7 gene RMs were screened by PCR and confirmed by Southern blot analysis. The AbSte7 RMs were screened by PCR with the combination of different primer pairs of P9/P10, P9/P11, and P12/P10 (Table 1). Southern blotting was performed using the DIG High Prime DNA Labeling and Detection Starter kit II (Roche, Germany) according to the manufacturer’s instructions. Genomic DNA was extracted with the E.Z.N.A. fungal DNA kit (Omega Bio-Tek, USA) and digested with SalI overnight. Hybridization was carried out using the DIG-labeled probe construct by PCR amplification with the primer pair p13 p14 and the genome of the AbSte7 RMs.

Genetic complementation was administered by co-transforming a 2.4 kb AbSte7 fragment under its native promoter with the pCB1532 plasmid carrying a sulfonyl-urea-resistant gene [19] into protoplasts of an AbSte7 mutant. The complementation transformants were selected with 5 μg/ml sulfonyl urea (Chem Service, Inc., USA) and tested for phenotypic restoration.

Growth Rate Assays

PDA, 1/4 PDA, and water/agar media were used for growth rate assays. Mycelia agar discs (5 mm diameter) were acquired from the periphery region of A. brassicicola WT, AbSte7 RMs, and the complementation strain grown on PDA media using a cork borer. The discs were transferred to new media of PDA, 1/4 PDA, and water/agar. The colony diameters were measured 6 days post inoculation (dpi).

Tolerance to Salt and Oxidative Stress

Mycelia agar discs (5 mm diameter) from the colonies of different strains grown on PDA were transferred onto new PDA plates containing different concentrations of salts or H2O2, and then incubated at 25℃ for 6 days. Radial growth of A. brassicicola WT, AbSte7 RMs, and complementation strains on the PDA plates were assessed by measuring the diameters of the colonies.

Measurement of the Amount of Fungal Melanin

The amount of melanin in A. brassicicola was measured according to the standard protocols [1], with slight modification. Mycelia grown on PDA plates at 25℃ for 10 days were harvested and used for melanin extraction. Briefly, mycelia were immersed in 2% NaOH (dilution coefficient = 1:10) and boiled at 100℃ for 2 h. The cocktail was then acidified to pH 2.0 with concentrated HCl to precipitate the melanin pigments. The resulting pigments were recovered by centrifugation at 6,000 ×g for 15 min and dissolved in 2% (dilution coefficient = 1:20) NaOH to measure the absorbance at 459 nm.

Fungal Pathogenicity

Fungal pathogenicity was evaluated using detached intact or wounded cabbage l eaves. PDA p lates of a ll fungal s trains were cultured for 6 days at 25℃. For inoculation, fresh leaves from 6-week-old B. rapa ssp. chinensis cv. wuyueman grown in the greenhouse were removed and placed on wet paper towels at the bottom of 90 mm Petri plates. Mycelia agar discs (5 mm diameter) acquired from the colonies of A. brassicicola WT, AbSte7 RMs, and the complementation strain grown on PDA plates were carefully transferred onto the detached cabbage leaves. After inoculation, the leaves were incubated in a mist chamber at 25℃ in darkness for lesion development.

 

Results

Isolation and Characteristics of MAPKK Genes of A. brassicicola

The genome sequence of A. brassicicola ATCC 96836 has been deposited at DDBJ/EMBL/GenBank under Accession No. ACIW00000000. Three contigs (ACIW01002982, ACIW01001143, and ACIW01000395) that harbored putative MAPKK genes were identified from this genome by blasting with the protein sequence of a MAPKK gene, Ste7, in yeast and its homolog in P. tritici-repentis. Among them, a 1,513 bp fragment contained the highest similarity to other MAPKKs in the pheromone response pathway. This genomic sequence was further confirmed by PCR using the genomic DNA of A. brassicicola WT abta14 as the template and Pf/Pr as the primer pair. RT-PCR was employed to amplify its corresponding cDNA sequence. Sequence alignment between the PCR product of genomic DNA and the cDNA fragment revealed that three introns (52, 55, and 50 nucleotides in length) are imbedded in the genomic DNA sequence of A. brassicicola. This gene was named AbSte7 (A. brassicicola Ste7), a putative yeast Ste7 homolog in A. brassicicola, and its functions were further characterized. Phylogenetic analysis with known MAPKKs in fungal species indicated that AbSte7 is most similar to its homolog of P. teres (Fig. 1).

Fig. 1.Fungal MAPKK phylogenetic tree based on amino acid sequences. MEGA 6.0 was used to generate the phylogenetic tree by the neighbor-joining method. Each sequence is presented with its NCBI accession number with two capital letters in brackets representing the original fungal species. AB: Alternaria brassicicola, BC: Botrytis cinerea, MO: Magnaporthe oryzae, FG: Fusarium graminearum, NC: Neurospora crassa, SS: Sclerotinia sclerotiorum, NH; Nectria haematococca, NF: Neosartorya fischeri, PT: Pyrenophora teres, MT: Myceliophthora thermophila.

Generation of AbSte7-Targeted Gene RMs

A gene disruption strategy was adopted to investigate the functions of AbSte7 in A. brassicicola. With this strategy, the coding region of AbSte7 was replaced with the HygB gene cassette (Fig. 2A). Fungal transformants were subjected to PCR to screen AbSte7 RMs (ΔAbSte7). As shown in Fig. 2B, the two transformants M1 (lane 1) and M2 (lane 4) produced a 4.9 kb amplicon with the outer primer pair P9/P10, consistent with the replacement of AbSte7 with the HygB gene cassette, whereas WT and ectopic transformants (lanes 2, 3, 5, and 6) produced amplicons of only 4.3 kb. When the disrupted mutants were further validated using two primer pairs, P9/P11 and P10/P12, the two transformants of M1 and M2 produced 1.48 kb and 1.59 kb fragments, respectively, as expected, whereas the WT strain abta14 and four other ectopic transformants produced no PCR product (Fig. 2B middle panel for P9/P11 primers; bottom panel for P10/P12 primers).

Fig. 2.Strategy for the gene disruption of AbSte7 and verification of the gene replacement transformants. (A) Production of the AbSte7 gene replacement construct by double-joint PCR. Relative primer locations are indicated on the schematic diagram. (B) Screening of AbSte7 knock-out mutants by PCR with the primer pairs of P9/P10 (top panel), P9/P11 (middle panel), and P10/P12 (bottom panel). M: DNA size marker; lanes 1–6: putative AbSte7 knock-out mutants; WT: A. brassicicola wild type. (C) Verification of the AbSte7 replacement mutants (RMs) M1 and M2 by Southern blotting. Genomic DNA was digested with ScaI and probed with the PCR product generated by primers P13/P14. M: DNA size marker; WT: A. brassicicola wild type; M1 and M2: two AbSte7 RMs; E1 and E2: two ectopic transformants.

Mutants of M1 and M2 were further verified based on disruption of AbSte7 by Southern blot analysis. Using a probe composed of about 1.0 kb PCR product amplified using the primer pair P13/14, a 2.1 kb hybridizing band was detected in the ΔAbSte7 mutants M1 and M2, respectively, whereas no band was observed in WT or the two ectopic transformants E1 and E2. These results indicated that only a single copy of the replacement vector had been integrated into the genome of the ΔAbSte7 mutants M1 and M2.

AbSte7 RMs Show Altered Vegetative Growth, Colony Morphology, and Conidiation

Growth assays were conducted on PDA, 1/4 PDA, and water agar media with the A. brassicicola WT strain, ΔAbSte7 mutants M1 and M2, and the complementation strain (C1). The AbSte7 RMs were consistently smaller in diameter than the WT strain or the complementation strain on all growth media (Fig. 3A). Furthermore, the colony morphology of the ΔAbSte7 mutants differed notably from the WT strain. On PDA plates, the WT strain was characteristically covered with thick black layers of spores and showed very little aerial mycelia. In comparison, grayish aerial mycelia were distributed over the entire colony surface in the ΔAbSte7 mutant plates (Fig. 3A). The complementation transformant of ΔAbSte7 mutant recovered the change in morphology induced by AbSte7 disruption, forming black layers of spores (Fig. 3A).

Fig. 3.Growth rate, hyphal morphology, and conidia formation of wild-type strain and AbSte7 replacement mutants (RMs). (A) Effect of AbSte7 replacement on the morphology and growth of A. brassicicola. Fungal growth was measured at 6 days after incubation on different culture plates. (B) Hyphal morphology of WT and M1. Pictures of (1) and (2) were taken from slides, whereas (3) and (4) were taken from PDA plates directly. (C) Formation of conidia by A. brassicicola WT and M1 strains grown on PDA. Boxes in the photograph (2) represent spores from the WT and M1 strains. WT: wild-type strain; M1 and M2: two AbSte7 RMs; C1: complementation strain of AbSte7 RM.

Microscopic observation showed that fungal hyphae of the AbSte7 disruptants contained coiled structures in some parts (Fig. 3B(1)). The hyphae septae of ΔAbSte7 mutants were shorter and thicker than those of the WT strain (Fig. 3B(2,3)). Some of the hyphae of AbSte7 disruptants were observed to have swollen segments (Fig. 3B(4)). Disruption of the AbSte7 gene also affected conidiation of A. brassicicola. The ΔAbSte7 M1 mutant generated conidiophores similar to the WT strain (Fig. 3C(1)), but the conidiophores could not produce fully developed conidia (Fig. 3C(2)).

AbSte7 Is Involved in Salt and Oxidative Stress

To assess the role of AbSte7 in response to oxidative and salt stress, A. brassicicola WT, ΔAbSte7 mutants M1 and M2, and the complementation strain were cultured on PDA plates containing different concentrations of salts or H2O2. The ΔAbSte7 mutants grew notably faster than the WT and complementation strain C1 on PDA containing sorbitol or sucrose, especially in the highest concentration of sugars (Fig. 4). When salt stress was induced by KCl, the ΔAbSte7 M1 and M2 displayed a slight increase in growth rate relative to A. brassicicola WT and the complementation strain. However, ΔAbSte7 mutants M1 and M2 showed significantly increased growth compared with WT or complementation strain C1 at high concentrations of NaCl. There was very small growth of WT or complementation strain C1 on PDA plates containing the high concentration of NaCl (1.5 M); however, ΔAbSte7 mutants M1 and M2 grew at the concentration of NaCl.

Fig. 4.Effect of AbSte7 replacement on the oxidative and salt stress tolerance of A. brassicicola. Photographs were taken at 6 days after incubation on potato dextrose agar (PDA) or PDA containing different concentrations of salts and H2O2. For the treatments of different kinds of salts, the left and right panels represent low (0.15 M) and high (1.5 M) concentrations of salts, respectively. The concentration of H2O2 was 5 mM (left) and 30 mM (right), respectively.

Disruption of AbSte7 resulted in increased sensitivity to oxidative stress (Fig. 4). AbSte7 RMs M1 and M2 showed no viability on PDA plates containing 30 mM H2O2, whereas WT and C1 could still survive and grow at this concentration of H2O2.

AbSte7 Is Necessary for Melanin Synthesis and Spore Compartments

Melanin plays an important role in spore development and abiotic resistance in Alternaria species; therefore, we examined the melanin production in the ΔAbSte7 mutants. In addition to the white color growth on PDA plates, which might have indicated less production of melanin, both ΔAbSte7 mutants M1 and M2 produced less melanin than the WT and C1 strains, with only 42.3% and 39.7% production being observed relative to the WT strain (Fig. 5A). Moreover, disruption of the AbSte7 gene reduced the number of spore compartments in A. brassicicola. Whereas an average of 6.9 compartments per spore was observed in the WT strain, only an average of 4.5 and 4.2 compartments per spore were counted in ΔAbSte7 M1 and M2, respectively (Fig. 5B).

Fig. 5.Quantification of melanin (A) and spore compartments (B) of WT, M1, M2, and C1. WT: wild-type strain; M1 and M2: two AbSte7 replacement mutants (RMs); C1: complementation strain of the AbSte7 RM. Each column represents the mean ± standard error of three independent experiments with three replicates. The statistical significance of differences between strains was determined by the Student’s t-test (p < 0.05).

Disruption of MAPKK AbSte7 Leads to a Dramatic Decrease in Fungal Virulence

To determine whether AbSte7 is essential for pathogenicity toward cabbage, we inoculated detached cabbage leaves with mycelial plugs of A. brassicicola WT, ΔAbSte7 mutants, and C1 strain. As shown in Fig. 3C(2), the conidia of AbSte7 RMs were not fully mature and therefore mycelial plugs of different strains were used as inoculum. Typical lesions appeared at 3 dpi on cabbage leaves inoculated with the WT and C1 strains, but not with the ΔAbSte7 mutants (Fig. 6, upper lanes).

Fig. 6.In vivo pathogenicity assay on detached cabbage leaves. Detached cabbage leaves were inoculated with the mycelial plugs from WT, M1, M2, and C1 strains of A. brassicicola. The inoculated leaves were then incubated in a mist chamber at 25℃ for lesion development. Upper panels: pathogenicity assay on intact leaves. Lower panels: pathogenicity assay on wounded leaves.

Successful infection processes involve penetration and colonization in host plants. Therefore, inoculation on wounded leaves was conducted to explicate the deficient steps in the pathogenicity of the ΔAbSte7 mutant. Accordingly, cabbage leaves were wounded with a sterilized needle before inoculation. As a result, lesions appeared on all inoculated leaves, but their rates of expansion differed greatly (Fig. 6, lower lane). On leaves inoculated with A. brassicicola WT and the complementation C1 strains, lesions continued to grow and eventually developed necrotic tissue (dead patches) (Fig. 6, lower lanes). The lesions on leaves inoculated with the ΔAbSte7 mutants M1 and M2 only appeared around the inoculation site and stopped expanding, even after prolonged incubation.

 

Discussion

The MAPK pathways are conserved signal transduction modules composed of three sequentially activated kinases, MAPK, MAPKK, and MAPKKK. There are different numbers of MAPKs, MAPKKs, and MAPKKKs in fungal species. For example, in Cryphonectria parasitica, at least six MAPKKKs, three MAPKKs, and four MAPKs were found [13]. The MAPKKK Ste11p of S. cerevisiae was involved in pheromone response, filamentous growth pathway, and high osmolarity/glycerol pathways [3]. Therefore, it is important and necessary to functionally analyze every protein kinase to understand the specific MAPKKK-MAPKK-MAPK interactions.

Our genome analysis of A. brassicicola revealed the presence of three MAPKK-related genes. Among them, contig-ACIW01002982 was named AbSte7 owing to its similarity to the yeast MAPKK Ste7 (Fig. 1). Prior to this study, the Fus3/Kss1 pathway was characterized in many different phytopathogens, including Aspergillus niger [14], A. alternata [10], Magnaporthe grisea [22], Bipolaris oryzae [12], and Fusarium graminearum [7]. However, these reports all focused on the Fus3 homologs and corresponding proteins, but the functions of MAPKKs and MAPKKKs in phytopathogenic fungal species have seldom been studied. Functional comparisons of the MAPK signaling pathway of Ste11-Ste7-Fus3 in S. cerevisiae indicate that there might be mediating roles of AbSte7 between AbSte11 and AbFus3 as the pathway AbSte11-AbSte7-AbFus3 in A. brassicicola.

To investigate the roles of the AbSte7 gene, a gene disruption and complementation strategy was adopted. The AbSte7 RMs showed reduced pathogenicity, which differed somewhat from that of the RM of Amk1, a MAPK gene of A. brassicicola. AbSte7 and Amk1 RMs had similar pathogenicity on intact leaves and different pathogenicity on wounded leaves. Whereas inoculation of wounded host leaves with Amk1 RMs resulted in restoration of pathogenicity [4], AbSte7 RMs f ormed v ery small lesions around the inoculation sites (Fig. 6, lower panels). Based on these results, the AbSte7 gene might confer more important roles in penetration ability and colonization ability than the Amk1 gene. When compared with the Amk1 gene, the AbSte7 gene as an upstream signaling component may have more contacting elements for pathogenicity in A. brassicicola. In particular, the incomplete development of conidia from the conidiophores of the ΔAbSte7 mutant indicates that the gene is strongly linked to the development of conidia. The underdeveloped conidia of the ΔAbSte7 mutant might be due to the conidiophores being non-functional, despite having normal appearance, or to developmental defect in the conidiophores as a result of the absence of AbSte7.

Melanin plays an important role in spore development and pathogenicity [11,23]. RMs of AbSte7 significantly decreased melanin production (Fig. 5A) and reduced spore compartments (Fig. 5B). These findings indicate that ΔAbSte7 mutants in A. brassicicola reduced the pathogenicity owing to their being less production of melanin and less formation of spores. Furthermore, the reduced melanin production can be linked to impaired tolerance to oxidative stress (Fig. 4). The RMs of AbSte7 were unable to grow on PDA plates containing 30 mM H2O2, strongly suggesting that the MAPK signaling pathway mediated by AbSte7 positively regulates oxidative stress response and plays a role in protecting A. brassicicola against severe oxidative stress. This differs from most other reported fungi such as Beauveria bassiana, Botrytis cinerea, B. oryzae, Candida albicans, Colletotrichum lagenarium, M. grisea, and Trichoderma harzianum, in which oxidative stress response is positively regulated by the Hog1 homolog pathway [20]. Moreover, phytopathogenic pathogens should detoxify reactive oxygen species effectively for successful infection of host [9], and the inability of ΔAbSte7 mutants to detoxify H2O2 may be partly responsible for their reduced pathogenicity on cabbage leaves.

Our results highlight the important role of AbSte7 in development, conidiation, melanin accumulation, and fungal virulence in A. brassicicola. To the best of our knowledge, this is the first report elucidating the functional roles of a MAPKK in A. brassicicola. The disruption and complementation of the AbSte7 gene strongly indicate that the gene plays an essential role in the pathogenicity of A. brassicicola by affecting melanin synthesis and penetration. These findings contribute to the overall understanding of the roles of different layers of genes in the MAPK signaling pathways involved in the pathogenicity of A. brassicicola.