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TeA is a key virulence factor for Alternaria alternata (Fr.) Keissler infection of its host
Plant Physiology and Biochemistry 115 (2017) 73e82
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Research article
TeA is a key virulence factor for Alternaria alternata (Fr.) Keissler infection of its host Ye Kang a, Hongwei Feng a, Jingxu Zhang a, Shiguo Chen a, Bernal E. Valverde a, b, Sheng Qiang a, * a b
Weed Research Laboratory of Nanjing Agricultural University, No.1 Weigang, Xuanwu District, Nanjing 210095, China n y Desarrollo en Agricultura Tropical, P.O.Box 2191, Alajuela 4050, Costa Rica Investigacio
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 November 2016 Received in revised form 27 February 2017 Accepted 2 March 2017 Available online 4 March 2017
A toxin-deficient mutant strain, HP001 mutant of Alternaria alternata, whose mycelium is unable to infect its host, produces little tenuazonic acid (TeA) toxin. How TeA plays a role in initiating host infection by A. alternata remains unclear. In this research we use Imaging-PAM based on chlorophyll fluorescence parameters and transmission electron microscopy to explore the role of TeA toxin during the infection process of A. alternata. Photosystem II damage began even before wild type mycelium infected the leaves of its host, croftonweed (Ageratina adenophora). Compared with the wild type, HP001 mutant produces morphologically different colonies, hyphae with thinner cell walls, has higher reactive oxygen species (ROS) content and lower peroxidase activity, and fails to form appressoria on the host surface. Adding TeA toxin allows the mutant to partially recover these characters and more closely resemble the wild type. Additionally, we found that the mutant is able to elicit disease symptoms when its mycelium is placed on leaves whose epidermis has been manually removed, which indicates that TeA may be determinant in the fungus recognition of its plant host. Lack of TeA toxin appears responsible for the loss of pathogenicity of the HP001 mutant. As a key virulence factor, TeA toxin not only damages the host plant but also is involved in maintaining ROS content, host recognition, inducing appressoria to infect the host and for allowing completion of the infection process. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Alternaria alternata Reactive oxygen species (ROS) Tenuazonic acid (TeA) Virulence factor
1. Introduction Alternaria alternata, one of the most cosmopolitan saprophytic fungal pathogens, also can infect numerous crops, causing yield and economic losses by at least 20% (Shafique et al., 2012). The mechanism of infection of A. alternata and the signaling pathways involved have been widely addressed (Chung, 2013; Akimitsu et al., 2014). Pathogenesis of A. alternata begins with host infection thus mutual recognition between host and pathogen is very important. Lectin, cutin, mannose glycoprotein, galactose and trehalose all can serve as signaling molecules that can be recognized by the host (Miyakawa et al., 2014; Poueymiro et al., 2014). Reactive oxygen species (ROS) produced by the host plant in the initial stage of infection can act as second messengers in activating an immune response (Heller and Tudzynski, 2011; Tudzynski et al., 2012).
* Corresponding author. E-mail address: [email protected] (S. Qiang). http://dx.doi.org/10.1016/j.plaphy.2017.03.002 0981-9428/© 2017 Elsevier Masson SAS. All rights reserved.
Consequently, the pathogen needs an effective antioxidant protection system to resist ROS from the host (Egan and Talbot, 2008; Samalova et al., 2014). ROS also regulate pathogenic fungal growth and differentiation; their balance is important during spore germination, appressorium development and hyphal tip growth (Egan et al., 2007; Guo et al., 2011; Kim et al., 2009). The response to ROS signal is regulated by the MAPK signaling pathway and/or some other pathways, which in concert also regulate the activity of peroxidases. In the tangerine pathotype of A. alternata, the Fus3/ Kss1 MAPK signaling pathway can singly or cooperatively regulate the pathogenicity with the AaAP1 gene (homologous transcription factor of YAP gene in Saccharomyces cerevisiae) that controls ROS levels in the pathogens (Lin et al., 2010). Thus ROS are key signaling molecules in pathogenicity. Upon infection, the pathogen forms appressoria that use turgor pressure generated by glycerol to penetrate the plant's epidermis (Howard et al., 1991). The pathogen destroys the host's cell wall by secreting cell wall degrading enzymes (Cho, 2015). Mycotoxins are common virulence factors in the host infection
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by plant pathogens, determining the interaction between the two organisms (Howlett, 2006). Most of the pathogens use toxins to kill plant cells obtaining nutrients from decaying tissue. Toxins could bind different targets, inhibit the activity of host plant enzymes and affect the biosynthesis of key metabolites. Some pathogens produce phytohormones that interfere with plant normal physiological processes and induce a ROS burst that damages host cells and tis€bius and Hertweck, 2009). Different toxins sues (Howlett, 2006; Mo have different modes of action. Toxins usually require photosensitizers to damage the host plant (Daub et al., 2005), bind target proteins (Chen et al., 2007, 2010), destroy biomembrane integrity (Abbas et al., 1994), deplete metal ions (Haas et al., 2008), block cell € bius and energy transfer (Meiss et al., 2008), induce apoptosis (Mo Hertweck, 2009; Stone et al., 2000), destroy the cytoskeleton € bius and Hertweck, 2009), and change gene expression (Mo € bius and Hertweck, 2009) to induce cell death. A remarkable (Mo non-host specific phytotoxin produced by A. alternata causing the brown leaf spot disease of croftonweed (Ageratina adenophora) is tenuazonic acid (TeA) (Qiang et al., 2010). TeA is a natural photosystem II inhibitor that blocks electron transport beyond QA by competing with QB for the QB-site in the D1 protein (Chen et al., 2007, 2010). TeA also inhibits the activity of chloroplast ATPase and Ferredoxin-NADP (þ) reductase (FNR). Oxidative damage resulting from TeA-induced ROS in chloroplasts of mesophyll cells is responsible for the TeA-triggered cell destruction and leaf necrosis (Chen et al., 2010). TeA besides damaging the host plant, may also effect A. alternata's pathogenicity by regulating ROS balance. But the mechanism by which TeA determines the infection of the host by A. alternata remains unclear. We here report studies on the mechanism of action of TeA in relation to pathogenicity using a toxin-deficient mutant strain.
2. Materials and methods 2.1. Biological materials and growth conditions A. alternata strain NEW was used as the reference, wild type; a toxin-deficient mutant strain, DHP001 obtained by restriction enzyme-mediated integration (REMI) is referred herein as the HP001 mutant strain. Both NEW and its derivative mutant were cultured on PSA medium (potato 200 g/L, sucrose 20 g/L, agar 15 g/ L) at 28 C. A. adenophora plants from seed were grown in the greenhouse (20e28 C, 70% humidity, 16/8 h). All foliage samples used in this study came from the upper second and third leaves of plants at the ten-leaf growth stage. Detached leaves were washed with ddH2O, placed on moistened filter paper and their petioles bundled with moistened cotton.
2.2. Monitoring the injury of A. adenophora leaves by A. alternata mycelia using Imaging-PAM Fungal mycelia were harvested from PS medium (potato 200 g/ L, sucrose 20 g/L) at 28 C, 120 rpm for 4e5 days. 0.1 g mycelia dried by filter paper were taken and mixed with 500 ml filtrates, then smashed using a homogenizer. An Imaging-PAM Chlorophyll Fluorometer (M Series, Heinz Walz GmbH, Germany) was used to measure photosynthesis after inoculating fungal mycelia onto A. adenophora leaves. The following recommended settings were applied in the study: pulse modulated measuring light intensity (ML) ¼ 2; measuring light pulse frequency (MF) ¼ 1; Gain ¼ 2; Damping ¼ 2; actinic light (AL) ¼ 8; and saturating flashes ¼ 10. The photosynthesis indices determined are presented in Table 1. 2.3. Effect of pathogen's filtrates on the pathogenicity of A. alternata mycelia A. adenophora leaves were inoculated with mycelium of the wild type and HP001 mutant alone and in combination with filtrates of each other. Photosynthetic indices were determined by ImagingPAM at 2, 6, 10, 14, and 18 h after inoculation and compared to those obtained from leaves inoculated with the wild type filtrate as control. The experiment was replicated three times. 2.4. Effect of TeA to the pathogenicity of A. alternata mycelium Wild type and HP001 mutant mycelium alone and in combination with 1000 ppm or 100 ppm TeA were inoculated onto A. adenophora leaves. The toxin applied alone at both concentrations was used as a control treatment. Photosynthetic indices were measured by Imaging-PAM as before. Also, HP001 mutant mycelium in combination with 0, 50, 60, 70, 80, 90 ppm TeA were inoculated onto A. adenophora leaves to determine if the recovery of pathogenicity of the HP001 mutant is TeA-concentration dependent. The experiment was replicated three times. 2.5. Effect of HP001 protein to the pathogenicity of A. alternata Wild type and HP001 mutant mycelia, HP001 mutant mycelium plus HP001 protein, and HP001 mutant mycelium plus vector pET28a were inoculated onto A. adenophora leaves and photosynthetic indices determined as before. 2.6. Microscopic observation of mycelium Mycelia of both the wild type and HP001 mutant and of HP001 mutant growing in presence of TeA were harvested from PS medium at 28 C, 120 rpm for 3 days and immobilized by
Table 1 Photosynthesis indices used in this study. Indexes Fo Fm Fv/Fm F Fm’ NPQ qN qP qL
Formulae
(Fm-Fo)/Fm
(Fm-Fm’)/Fm’ (Fm-Fm’)/(Fm-Fo’) (Fm’-F)/(Fm’-Fo’) (Fm’-F)/(Fm’-Fo’) Fo’/F ¼ qP Fo’/F
Descriptors Base fluorescence Maximum fluorescence after dark adaptation Maximal photochemical efficiency of PSII Current fluorescence yield in the presence of actinic illumination Maximal fluorescence yield Non-photochemical quenching Total non-photochemical quenching Photochemical quenching Photochemical quenching (Kramer Lake Model)
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glutaraldehyde to observe their cell wall characteristics by transmission electron microscopy (Hitachi H-7560). Fungal mycelia were also stained with aniline blue after inoculation onto A. adenophora leaves to observe the evolution of infection in the light microscope. The experiment was replicated three times. Mycelia were also inoculated onto detached onion epidermis, instead of A. adenophora epidermis that is difficult to remove from the leaves, for 24 h to observe and photograph the appressorium formation in light microscope. 2.7. ROS detection Intracellular ROS levels were monitored in mycelia using nitroblue tetrazolium (NBT) as described by Guo et al. (2011). Superoxide production was observed in light microscope and measured the gray scale of the light micrographs. The experiment was replicated three times. 2.8. Effect of epidermis removal on the pathogenicity of A. alternata The epidermis of A. adenophora leaves was manually removed using tweezers before inoculating HP001 mutant mycelium onto the subjacent leaf tissue for 24 h to observe its pathogenicity and detect the ROS content. The experiment was replicated three times. 2.9. Peroxidase enzyme activity Mycelia were ground under liquid nitrogen and suspended in PBS buffer as crude enzyme suspension. Laccase and ascorbate peroxidase activities were measured as described by Shelp et al. (1999) and Nakano and Asada (1981), respectively. Total glutathione peroxidase activity was measured according to a commercial protocol (Beyotime Biotechnology, S0058) and activity of ligninase was measured according to Faison and Kirk (1985). These tests were done at least three times, each with three replications. 2.10. Fus3 gene expression analyses Total RNA was extracted from mycelia growing in PS medium (28 C, 120 rpm for 1e7 days) and at 4-h intervals from previously inoculated A. adenophora leaves for 2e18 h to determine the expression levels of fus3. Changes during fungal development and infectious growth were calculated as 2-△△Ct, where △△Ct ¼ (Ct, target gene - Ct, actin gene)test condition - (Ct, WT - Ct, actin gene). qRT-PCR was performed with three independent pools of tissues in three sets of experimental replicates using the following primer pairs: actin_F: 50 -ggcaacattgtcatggtatg-3’; actin_R: 50 -gaggaagcaagaatggaac-3’; fus3_F: 50 -taccgagcacatcgagaa-3’; fus3_R: 50 -tcccaggaaggactacc0 3 3. Results 3.1. Photosynthetic system of the host plant was damaged before infection by A. alternata
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not penetrated the foliar tissue (Fig.1ABCD). Ten hours after inoculation, the foliar region inoculated with the wild type had begun developing lesions (Fig. 1D) and qL and qP and NPQ further decreased (Fig. 1A); the hyphae of the wild type were already colonizing the leaves and obvious disease spots developed by 14 h after inoculation (Fig. 1D). At this time, both the photochemical and non-photochemical quenching coefficients decreased drastically; quantum yield Y (II) of PSII also decreased (Fig. 1A). At 18 h after inoculation, disease spots continued to expand and the photosystem II reaction center became seriously damaged. In contrast, the regions inoculated with mutant still lacked disease spots and the chlorophyll fluorescence indexes remained at almost normal levels after 18 h (Fig. 1ABCD). The chlorophyll fluorescence indexes changed earlier than disease spots appeared indicating that active substances produced by hyphae could react early and cause photosynthetic system damage before the hyphae actually infected the host. Further experiments were conducted to determine if indeed the TeA toxin was involved in the onset of tissue damage and pathogen infection. 3.2. TeA toxin is essential for aerial hyphal growth The wild type fungus formed compact mycelium masses in liquid medium, in contrast to the sparse ones formed by the mutant. However, addition of TeA to the liquid PS medium restored the morphology of the mutant mycelia to closely resemble that of the wild type (Fig. 2A). Therefore TeA apparently contributes to maintaining mycelium morphology. 3.3. TeA toxin restores cell wall thickness Transmission electron micrographs revealed that the cell wall of the mutant was significantly thinner (0.07 mm) than that of the wild type (0.3 mm). By adding TeA, the cell wall of the mutant could be restored to half of the normal thickness (Fig. 2B and C). 3.4. Filtrates of cultured wild type mycelium partially restore the pathogenicity of the mutant The chlorophyll fluorescence index of leaf areas inoculated only with mutant hyphae, wild type culture filtrates, or wild type culture filtrates previously sterilized at high temperature had no significant variation 18 h after inoculation (Fig. 3). However, the chlorophyll fluorescence indexes of leaf sections inoculated with the mutant mycelium supplemented with a wild type culture filtrate, were similar to those in foliar areas inoculated by the wild type, regardless of high-temperature sterilization of the filtrate. Compared with 2 h after inoculation, these inoculated areas had significantly increased Y(NO), and significantly decreased NPQ and qN, at 18 h after inoculation (Fig. 3). The active ingredient in wild type culture filtrates, likely to be TeA, could make the mutant exhibit similar pathogenicity to that of the wild type, and the active ingredient is unlikely to be a large molecule such as a protein since it was not labile at high-temperature (data not show). 3.5. TeA toxin partially restores the pathogenicity of the mutant
Damage to the photosynthetic system of A. adenophora leaves inoculated with the wild type and mutant strains was observed by Imaging-PAM. Two hours after inoculation with either strain, no disease symptoms were observed in the inoculated regions, which also had normal chlorophyll fluorescence indexes (Fig.1ABC). Six hours after inoculation, disease spots were still absent, but the photochemical quenching coefficient (qL and qP) and the nonphotochemical quenching (NPQ) of the region inoculated with the wild type mycelium had decreased although its hyphae still had
The chlorophyll fluorescence indexes of leaf areas inoculated with the wild type were similar to those measured when the foliage was inoculated with mutant supplemented with the TeA toxin at either 1000 ppm or 100 ppm, whereas the Y(NO) significantly increased, and the NPQ and qN significantly decreased at 18 h after inoculation compared with at 2 h. This is a clear indication that the PSII reaction center of the plant was blocked and that photodynamic damage occurred because of the inability of the
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Fig. 1. Fluorescence image of Alternaria alternata hyphae at the infection stage. A. The chlorophyll fluorescence indexes of Ageratina adenophora leaves from time of hyphal inoculation; B. Light photographs and C. Fluorescence images (Fv/Fm) of A. adenophora leaves inoculated by wild type (left circle) and HP001 mutant (△HP001, right circle) mycelia showing the development of lesions; D. Light micrographs of aniline-blue stained wild type mycelia growing in A. adenophora leaves. Bars equal 30 mm.
photosynthetic apparatus to dissipate excess light energy. The chlorophyll fluorescence indexes in the areas of leaves inoculated with the mutant or treated only with either 1000 ppm or 100 ppm TeA toxin did not change during the observation period, i.e. from two to 18 h after inoculation (Fig. 4) (The results of 1000 ppm TeA toxin treatment were confirmed in a separate bioassay in Fig. S1). Hyphae of the mutant mixed with TeA toxin at concentrations ranging from 50 to 100 ppm were inoculated on leaves to study the concentration-dependent effect of the toxin on pathogenicity. Damage to the photosynthetic apparatus was detected at 24 h after inoculation only after the addition of the highest concentration of 100 ppm (Fig. S2). Moreover, the addition of HP001 expression product had no effect on pathogenicity (Fig. S3). These results further point to TeA toxin likely being the biologically active ingredient in wild type culture filtrates, thus suggesting an important role of the toxin on pathogenicity.
3.6. TeA toxin promotes appressorium formation Hyphae of the wild type and mutant inoculated on onion epidermis grew normally, but those of the mutant failed to form appressoria (Fig. 5A and B). The wild type hyphae were difficult to remove manually from the onion epidermis at 24 h after inoculation but the mutant hyphae easily detached at this time because they lacked appressoria to attach them to the epidermis. However, mutant hyphae formed some appressoria structures when TeA toxin was added (Fig. 5C and D). This might be a reason for the
mutant partial recovery of pathogenicity by addition of TeA toxin.
3.7. TeA reduces the ROS content of the HP001 mutant The hyphal tip of the mutant contained about four times more ROS than that of the wild type, regardless if cultured on PSA medium or on a wet slide surface (Fig. S4). When infecting onion epidermis and A. adenophora leaves, the mutant contained seven to five times more ROS that the wild strain (Figs. 6 and 7A). The ROS concentration of mutant was reduced to wild-type levels during the infection process after adding 100 ppm TeA (Figs. 6A and 7A); the lowest concentration tested (50 ppm TeA) was unable to elicit this effect (Fig. 7A).
3.8. TeA toxin increases peroxidase activity of the HP001 mutant The peroxidase activity, including laccase, total glutathione peroxidase, ligninase, and ascorbate peroxidase, of both the wild type and mutant increased throughout the experimental period. But the peroxidase activity of the wild type was significantly higher than that of the mutant (Fig. 8ABCD). Adding TeA significantly enhanced the activity of laccase, ascorbate peroxidase, and total glutathione peroxidase of the mutant during the hyphal growing period, but not to the same level found in the wild type (Fig. 8ABC). The ligninase activity of the mutant was not affected by the addition of TeA (Fig. 8D).
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Fig. 2. Phenotype of growing mycelia and cell wall of Alternaria alternata. A. Phenotype of mycelia growing in liquid medium; B. Transmission electron micrographs of cross section of mycelium showing the cell wall. Bars equal 1 mm; C. Thickness of cell wall. Each column represents the mean ± the standard error from three independent experiments, each with at least three replicates. Letters above error bars indicate significance at P < 0.05.
Fig. 3. Contrast analyses of chlorophyll fluorescence parameters of wild type culture filtrate interacting with the HP001 mutant. 1. Wild type hyphae; 2. Mutant hyphae; 3. Mutant hyphae þ wild type culture filtrate (without autoclaving); 4. Mutant hyphae þ wild type culture filtrate (autoclaved); 5. Wild type culture filtrate (not-autoclaved); 6. Wild type culture filtrate (autoclaved). Each column represents the mean value ± the standard error from three independent experiments, each with at least three replicates. Columns labeled with the same letter are not significantly different (P < 0.05). Fluorescence images are color coded according to the patterns shown next to the images. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Contrast analyses of chlorophyll fluorescence parameters of TeA interacting with the HP001 mutant. 1. Wild type hyphae; 2. Mutant hyphae; 3. Mutant hyphae þ1000 ppm TeA; 4. Mutant hyphae þ100 ppm TeA; 5.1000 ppm TeA; 6.100 ppm TeA. Each column represents the mean value ± the standard error from three independent experiments, each with at least three replicates. Columns labeled with the same letter are not significantly different (P < 0.05). Fluorescence images are color coded according to the patterns shown next to the images. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Light micrographs of Alternaria alternata wild type and HP001 mutant hyphae infecting the epidermis of onion. A., B. Wild type and mutant hyphae infecting the epidermis of onion, respectively; C, D. Infection of leaf epidermis by mutant hyphae to which TeA toxin was added. Arrows point to appressoria. Bars equal 50 mm.
3.9. Leaves without their epidermis reduce the ROS content of HP001 mutant When the mutant mycelia were inoculated onto host leaves areas without epidermis, the ROS of mutant was reduced to about a half (Figs. S5A and B). This further suggests the involvement of TeA
in the fungus recognition of the host epidermis. 3.10. HP001 mutant infects host leaves whose epidermis has been removed The mutant caused disease when it was inoculated on leaves
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Fig. 6. Detection in light micrographs of ROS in the tip of hyphae of wild type and HP001 mutant (△HP001) under the epidermis of onion. A. Light micrographs of the superoxide in wild type and HP001 mutant under epidermis of onion. Bars equal 50 mm; B. Measurement of ROS content by the gray scale of the photomicrographs. Each column represents the mean ± the standard error from three independent experiments, each with at least three replicates. Columns labeled with the same letter are not significantly different (P < 0.05).
Fig. 7. Detection in light micrographs of ROS and illustration of lesions on Ageratina adenophora after addition of TeA at different concentrations. A. Photomicrographs of superoxide in photomicrographs of wild type, HP001 mutant (△HP001) and HP001 mutant under two concentrations of TeA. Bars equal 10 mm; B. Measurement of ROS content by the gray scale of the photographs. Each column represents the mean ± the standard error from three independent experiments, each with at least three replicates. Columns labeled with the same letter are not significantly different (P < 0.05). C. Lesions caused by HP001 mutant in combination with TeA at two concentrations of TeA compared with the wild type and TeA by itself (according to labels above circles).
whose epidermis had been previously removed (Fig. S5C). Evidently the epidermis is a major barrier for mutant infection but not for the wild type. This indicates that TeA may play a role in recognizing the host and breaking through the epidermis barrier. 3.11. TeA does not affect the Fus3/Kss1 MAPK signaling pathway Based on qRT-PCR, the wild type had significantly higher expression of the fus3 gene than the mutant, regardless of the stage of hyphal growth or hyphal infection of A. adenophora leaves. At the hyphal growth stage, the gene expression level peaked on the sixth day (Fig. 9A) and during the hyphal infection stage, the expression level reached peak at the 14th h (Fig. 9B). However, the expression level of the gene was not substantially improved by adding TeA to
the mutant. 4. Discussion Our lab has systematically evaluated the herbicidal activity of TeA to assess its potential as a bio-herbicide (Qiang et al., 2010). TeA was found to be a novel photosystem II inhibitor (Chen et al., 2007, 2010) whose phytotoxic action is mediated by the generation of singlet oxygen (Chen et al., 2015). In this study, we established the mechanism by which TeA determines the infection of the host by A. alternata and its role as a key virulence factor of the pathogen. The first step of the infection process is the mutual recognition of host plant and pathogen, which could regulate mycelial growth and appressorium formation (Aguirre et al., 2005; Lalucque and
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Fig. 8. Determination of peroxidase activity in wild type, HP001 mutant (△HP001) and the HP001 mutant after addition of TeA (DHP001 þ TeA) during a six-day growing period of Alternaria alternata. A. Laccase; B. Ascorbate peroxidase; C. Glutathione peroxidase; D. Ligninase. Each column represents the mean value ± the standard error from three independent experiments, each with at least three replicates. Columns labeled with the same letter are not significantly different (P < 0.05).
Fig. 9. Quantitative gene-expression analysis of fus3 gene among the wild type strain (WT), the HP001 mutant (DHP001), and the HP001 mutant after addition of TeA (DHP001 þ TeA) at the growing (A) and infection stages (B) of Alternaria alternata. Error bars represent the standard deviations.
Silar, 2003). ROS can function as molecular signals to regulate fungal sexual reproduction, growth, development, infection, and pathogenicity (Egan et al., 2007; Sun et al., 2006). Thus the ROS balance is a determinant of pathogenicity (Aguirre et al., 2005; Egan et al., 2007; Guo et al., 2011; Kim et al., 2009; Tanaka et al., 2006), and a complete peroxidase system in the fungus degrading excessive ROS would relieve damage to its cellular structure (Foley et al., 2016). An excessive accumulation of ROS in the conidia of A. brassicicola breaks the intracellular redox dynamic balance, ultimately
resulting in loss of pathogenicity (Kim et al., 2009). Results of this study show that the HP001 mutant recovers its colony morphology, reduces the ROS content in mycelia, increases the activity of peroxidase and recovers the pathogenicity after addition of exogenous TeA toxin. According to our results the apparent reason for the loss of pathogenicity in the mutant is the breaking of the ROS balance. Previous studies showed that a burst of ROS could be used as a signal during pathogen infection (Egan et al., 2007). But the content of ROS in the mutant is much more than that needed during the interaction between pathogen and host plant, with the
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disruption of ROS homeostasis being responsible for the loss of pathogenicity. Exogenous betaine and CaCl2 increase the activity of antioxidant enzymes that scavenge excessive ROS in Cystofilobasidium infirmominiatum and Debaryomyces hansenii, which could be conducive to improve the vitality of the fungi (An et al., 2012; Liu et al., 2011). Similarly, our research supports the notion that TeA could improve the peroxidase activity of the mutant and the scavenging of excessive ROS that would allow it recovering the ability to respond to the exogenous signals and recover its pathogenicity. Infection structure development is the key step in the colonization of the host plant by A. alternata after signal recognition. The formation of the appressorium is related to the molecular recognition interaction between the pathogen and its host (Deising et al., 2000; Ryder and Talbot, 2015). In M. oryzae, the formation of the appressorium is mainly related to the three signal transduction pathways, cAMP signaling pathway, MAPK signaling pathway and Ca2þ signaling pathway (Dean, 1997; Xu, 2000). Among them, the PMK1 signaling pathway (homologous with the yeast Fus3/Kss1 signaling pathway) is closely related to the formation and maturation of the appressorium. PMK1 was highly expressed during the formation of the appressorium, and a PMK1 mutant was unable to form it (Xu, 2000). Mst50, mst11, and mst7 located upstream of the PMK1 pathway all play an important role in the formation of the appressorium (Zhang et al., 2016; Zhao et al., 2005). The signaling pathway and some cooperating pathways regulate the response of fungi to ROS, and also the activity of a series of peroxidases in fungi (Lin et al., 2010). In our study, the mutant hyphae grew normally on the surface of host leaves, but could not infect them, mainly because its failure to form the appressorium. Excessive ROS in mycelial cell of the HP001 mutant may hinder the HP001 mutant to actuate the signaling pathway for the formation of the appressorium through the molecular recognition interaction between the pathogen and its host which was mediated by ROS. The mutant could recover the ability to form appressorium, infect host leaves and damage them after adding exogenous TeA. TeA could recover the mutant's responses to the exogenous ROS-mediated signals for the formation of the appressorium through improving the peroxidase activity and the scavenging of excessive ROS. This suggested that TeA toxin may be directly involved in the formation of appressorium during the infection process. Although the mutant lost its ability to infect the host, its hyphae could infect and colonize host leaf areas devoid of epidermis and thus it exhibited some pathogenicity. We posit that TeA toxin may play an important role in the hyphae entering into the mesophyll cell successfully at a primary infection stage. Last, the hyphae complete the infection stage and produce the characteristic disease spots. Being generally saprophytic, the fungi could kill host cells using toxins, obtain nutrients from the host plant and cause disease spots. In this research, the photosynthetic apparatus began degrading when wild type hyphae were inoculated on host leaves for 6 h, and excess light had damaged the photosynthetic apparatus. At the same time, hyphae did not infect the host 6 h after inoculation, thus disease spots maybe caused by some secondary metabolites. As it is difficult to detect these secondary metabolites, it was not possible to determine which one could cause damage. The ability of the mutant to infect host leaves devoid of epidermis clearly shows that the epidermis is a major barrier for the mutant that otherwise could infect its host and cause disease provided that the hyphae enter the mesophyll cells. We speculate that the restoration of pathogenicity of the mutant by the addition of TeA is an indication that this toxin begins damaging PSII before hyphae actually infects the host. Lesions would then be the result of both the action of the toxin and hyphae. Furthermore, after successful colonization of the host, TeA toxin may also help the
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pathogen in killing host cells, whose contents serve as nutrients to the pathogen thus facilitating the expansion of infection. It remains to verify this experimentally. The Fus3/Kss1 MAPK signaling pathway is the most widely studied MAPK signaling pathway in phytopathogenic fungi. Several Fus3/Kss1 homologous genes have been isolated that are also involved in sporulation, vegetative growth, resistance to oxidative stress, and melanin metabolism (Xu, 2000). Although the function of the Fus3/Kss1 MAPK signaling pathway is conserved, its role in several fungi is slightly different. In Alternaria spp., the Aafus3 gene has many functions, including in spore development, fungal infection, melanin biosynthesis, and pathogenicity (Lin et al., 2010). To further explore the molecular mechanism of the partial recovery of pathogenicity after adding TeA, we determined the expression of fus3 genes in the fungus’ signaling pathway. The expression of the fus3 gene was not significantly increased after adding TeA to the mutant. Since addition of the TeA toxin partially restored the cell wall thickness, appressorium formation and the pathogenicity of the HP001 mutant, we surmise that the metabolic pathway of TeA toxin that is involved in pathogenicity may be located downstream of the MAPK signaling pathway. It may regulate physiological processes like ROS metabolism, as well as appressorium and cell wall formation and pathogenicity, combined with MAPK signaling pathway, but the specific signaling networks need to be further studied. We can summarize the infection process of A. alternata. The phytopathogenic fungus starts secreting TeA toxin before infecting its host. The TeA toxin inhibits host photosynthetic electron transport in PSII leading to a ROS burst and damage of the epidermal and mesophyll cells. The hyphae forms appressoria after identifying the host epidermis then breaks this epidermal barrier, infecting and colonizing the host tissues to complete the infection process. The mutant produces insufficient TeA to damage the epidermis of the host, and has a high ROS content in the hypha cell, which causes the pathogen to be unable to respond to the host signal, failing to form the appressorium required to infect the host, and thereby losing pathogenicity. Thus TeA toxin is the key virulence factor for A. alternata infection of its host. Contributions Sheng Qiang (SQ) designed the study. Ye Kang (YK), Hongwei Feng (HF), Jingxu Zhang (JZ) and Shiguo Chen (SC) performed synthesis experiments and characterization. YK, JZ and SQ interpreted the data and drafted the manuscript. SQ, YK and Bernal E. Valverde (BV) majorly revised the manuscript. All authors contributed to and approved the final manuscript. Acknowledgements This work was supported by National Technology Research and Development Program of China and Science and Technology Pillar Program of Jiangsu Province [BE2014397]. The TeA toxin was kindly provided by Professor Chunlong Yang from the College of Sciences of Nanjing Agricultural University. The authors also gratefully thank Min Zhang, Qin Yao and Tingting Ran from the College of Life Science of Nanjing Agricultural University for their help to the experiments. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2017.03.002.
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References Abbas, H.K., Tanaka, T., Duke, S.O., Porter, J.K., Wray, E.M., Hodges, L., Sessions, A.E., Wang, E., Merrill Jr., A.H., Riley, R.T., 1994. Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases. Plant Physiol. 106, 1085e1093. Aguirre, J., Ríos-Momberg, M., Hewitt, D., Hansberg, W., 2005. Reactive oxygen species and development in microbial eukaryotes. Trends Microbiol. 13, 111e118. Akimitsu, K., Tsuge, T., Kodama, M., Yamamoto, M., Otani, H., 2014. Alternaria hostselective toxins: determinant factors of plant disease. J. Gen. Plant Pathol. 80, 109e122. An, B., Li, B., Qin, G., Tian, S., 2012. Exogenous calcium improves viability of biocontrol yeasts under heat stress by reducing ROS accumulation and oxidative damage of cellular protein. Curr. Microbiol. 65, 122e127. Chen, S., Kim, C., Lee, J.M., Lee, H.A., Fei, Z., Wang, L., Apel, K., 2015. Blocking the QBbinding site of photosystem II by tenuazonic acid, a non-host-specific toxin of Alternaria alternata, activates singlet oxygen-mediated and EXECUTERdependent signalling in Arabidopsis. Plant Cell Environ. 38, 1069e1080. Chen, S., Xu, X., Dai, X., Yang, C., Qiang, S., 2007. Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in QB-site of Chlamydomonas reinhardtii. BBA-Bioenerg. 1767, 306e318. Chen, S., Yin, C., Qiang, S., Zhou, F., Dai, X., 2010. Chloroplastic oxidative burst induced by tenuazonic acid, a natural photosynthesis inhibitor, triggers cell necrosis in Eupatorium adenophorum Spreng. BBA-Bioenerg. 1797, 391e405. Cho, Y., 2015. How the necrotrophic fungus Alternaria brassicicola kills plant cells remains an enigma. Eukaryot. Cell 14, 335e344. Chung, K.R., 2013. Mitogen-activated protein kinase signaling pathways of the tangerine pathotype of Alternaria alternata. MAP Kinase 2, 4. Daub, M.E., Herrero, S., Chung, K.R., 2005. Photoactivated perylenequinone toxins in fungal pathogenesis of plants. FEMS Microbiol. Lett. 252, 197e206. Dean, R.A., 1997. Signal pathways and appressorium morphogenesis. Annu. Rev. Phytopathol 35, 211e234. Deising, H.B., Werner, S., Wernitz, M., 2000. The role of fungal appressoriain plant infection. Microbes Infect. 2, 1631e1641. Egan, M.J., Talbot, N.J., 2008. Genomes, free radicals and plant cell invasion: recent developments in plant pathogenic fungi. Curr. Opin. Plant Boil 11, 367e372. Egan, M.J., Wang, Z.Y., Jones, M.A., Smirnoff, N., Talbot, N.J., 2007. Generation of reactive oxygen species by fungal NADPH oxidases is required for rice blast disease. P. Natl. Acad. Sci. U. S. A. 104, 11772e11777. Faison, B.D., Kirk, T.K., 1985. Factors involved in the regulation of a ligninase activity in Phanerochaete chrysosporium. Appl. Environ. Microb. 49, 299e304. Foley, R.C., Kidd, B.N., Hane, J.K., Anderson, J.P., Singh, K.B., 2016. Reactive oxygen species play a role in the infection of the necrotrophic fungi, Rhizoctonia solani in wheat. PLoS One 11, e0152548. Guo, M., Chen, Y., Du, Y., Dong, Y., Guo, W., Zhai, S., Zhang, H., Dong, S., Zhang, Z., Wang, Y., Wang, P., Zheng, X., 2011. The bZIP transcription factor MoAP1 mediates the oxidative stress response and is critical for pathogenicity of the rice blast fungus Magnaporthe oryzae. PLoS Pathog. 7, e1001302. Haas, H., Eisendle, M., Turgeon, B.G., 2008. Siderophores in fungal physiology and virulence. Annu. Rev. Phytopathol. 46, 149e187. Heller, J., Tudzynski, P., 2011. Reactive oxygen species in phytopathogenic fungi: signaling, development, and disease. Annu. Rev. Phytopathol. 49, 369e390. Howard, R.J., Ferrari, M.A., Roach, D.H., Money, N.P., 1991. Penetration of hard substrates by a fungus employing enormous turgor pressures. P. Natl. Acad. Sci. U. S. A. 88, 11281e11284. Howlett, B.J., 2006. Secondary metabolite toxins and nutrition of plant pathogenic fungi. Curr. Opin. Plant Biol. 9, 371e375. Kim, K.H., Willger, S.D., Park, S.W., Puttikamonkul, S., Grahl, N., Cho, Y.,
Mukhopadhyay, B., Cramer, R., Lawrence, C.B., 2009. TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen. PLoS Pathog. 5, e1000653. Lalucque, H., Silar, P., 2003. NADPH oxidase: an enzyme for multicellularity? Trends Microbiol. 11, 9e12. Lin, C.H., Yang, S.L., Wang, N.Y., Chung, K.R., 2010. The FUS3 MAPK signaling pathway of the citrus pathogen Alternaria alternata functions independently or cooperatively with the fungal redox-responsive AP1 regulator for diverse developmental, physiological and pathogenic processes. Fungal Genet. Biol. 47, 381e391. Liu, J., Wisniewski, M., Droby, S., Vero, S., Tian, S., Hershkovitz, V., 2011. Glycine betaine improves oxidative stress tolerance and biocontrol efficacy of the antagonistic yeast Cystofilobasidium infirmominiatum. Int. J. Food Microbiol. 146, 76e83. Meiss, E., Konno, H., Groth, G., Hisabori, T., 2008. Molecular processes of inhibition and stimulation of ATP synthase caused by the phytotoxin tentoxin. J. Biol. Chem. 283, 24594e24599. Miyakawa, T., Hatano, K., Miyauchi, Y., Suwa, Y.I., Sawano, Y., Tanokura, M., 2014. A secreted protein with plant-specific cysteine-rich motif functions as a mannose-binding lectin that exhibits antifungal activity. Plant Physiol. 166, 766e778. €bius, N., Hertweck, C., 2009. Fungal phytotoxins as mediators of virulence. Curr. Mo Opin. Plant Biol. 12, 390e398. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867e880. , A.C., François, J.M., Parrou, J.L., Peeters, N., Genin, S.A., 2014. Poueymiro, M., Cazale Ralstonia solanacearum Type III effector directs the production of the plant signal metabolite trehalose-6-phosphate. mBio 5 e02065e14. Qiang, S., Wang, L., Wei, R., Zhou, B., Chen, S., Zhu, Y., Dong, Y., An, C., 2010. Bioassay of the herbicidal activity of AAC-toxin produced by Alternaria alternata isolated from Ageratina adenophora. Weed Technol. 24, 197e201. Ryder, L.S., Talbot, N.J., 2015. Regulation of appressorium development in pathogenic fungi. Curr. Opin. Plant Biol. 26, 8e13. Samalova, M., Meyer, A.J., Gurr, S.J., Fricker, M.D., 2014. Robust anti-oxidant defences in the rice blast fungus Magnaporthe oryzae confer tolerance to the host oxidative burst. New Phytol. 201, 556e573. Shafique, S., Majeed, R.A., Shafique, S., 2012. Cymbopogon citrates: a remedy to control selected Alternaria species. J. Med. Plants Res. 6, 1879e1885. Shelp, B.J., Bown, A.W., McLean, M.D., 1999. Metabolism and functions of gammaaminobutyric acid. Trends Plant Sci. 4, 446e452. Stone, J.M., Heard, J.E., Asai, T., Ausubel, F.M., 2000. Simulation of fungal-mediated cell death by Fumonisin B1 and selection of Fumonisin B1-resistant (fbr) Arabidopsis mutants. Plant Cell 12, 1811e1822. Sun, C.B., Suresh, A., Deng, Y.Z., Naqvi, N.I., 2006. A multidrug resistance transporter in Magnaporthe is required for host penetration and for survival during oxidative stress. Plant Cell 18, 3686e3705. Tanaka, A., Christensen, M.J., Takemoto, D., Park, P., Scott, B., 2006. Reactive oxygen species play a role in regulating a fungus-perennial ryegrass mutualistic interaction. Plant Cell 18, 1052e1066. Tudzynski, P., Heller, J., Siegmund, U., 2012. Reactive oxygen species generation in fungal development and pathogenesis. Curr. Opin. Microbiol. 15, 653e659. Xu, J.R., 2000. MAP kinases in fungal pathogens. Fungal Genet. Biol. 31, 137e152. Zhang, S., Jiang, C., Zhang, Q., Qi, L., Li, C., Xu, J.R., 2016. Thioredoxins are involved in the activation of the PMK1 MAP kinase pathway during appressorium penetration and invasive growth in Magnaporthe oryzae. Environ. Microbiol. http:// dx.doi.org/10.1111/1462-2920.13315. Zhao, X., Kim, Y., Park, G., Xu, J.R., 2005. A mitogen-activated protein kinase cascade regulating infection-related morphogenesis in Magnaporthe grisea. Plant Cell 17, 1317e1329.
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Research article
TeA is a key virulence factor for Alternaria alternata (Fr.) Keissler infection of its host Ye Kang a, Hongwei Feng a, Jingxu Zhang a, Shiguo Chen a, Bernal E. Valverde a, b, Sheng Qiang a, * a b
Weed Research Laboratory of Nanjing Agricultural University, No.1 Weigang, Xuanwu District, Nanjing 210095, China n y Desarrollo en Agricultura Tropical, P.O.Box 2191, Alajuela 4050, Costa Rica Investigacio
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 November 2016 Received in revised form 27 February 2017 Accepted 2 March 2017 Available online 4 March 2017
A toxin-deficient mutant strain, HP001 mutant of Alternaria alternata, whose mycelium is unable to infect its host, produces little tenuazonic acid (TeA) toxin. How TeA plays a role in initiating host infection by A. alternata remains unclear. In this research we use Imaging-PAM based on chlorophyll fluorescence parameters and transmission electron microscopy to explore the role of TeA toxin during the infection process of A. alternata. Photosystem II damage began even before wild type mycelium infected the leaves of its host, croftonweed (Ageratina adenophora). Compared with the wild type, HP001 mutant produces morphologically different colonies, hyphae with thinner cell walls, has higher reactive oxygen species (ROS) content and lower peroxidase activity, and fails to form appressoria on the host surface. Adding TeA toxin allows the mutant to partially recover these characters and more closely resemble the wild type. Additionally, we found that the mutant is able to elicit disease symptoms when its mycelium is placed on leaves whose epidermis has been manually removed, which indicates that TeA may be determinant in the fungus recognition of its plant host. Lack of TeA toxin appears responsible for the loss of pathogenicity of the HP001 mutant. As a key virulence factor, TeA toxin not only damages the host plant but also is involved in maintaining ROS content, host recognition, inducing appressoria to infect the host and for allowing completion of the infection process. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Alternaria alternata Reactive oxygen species (ROS) Tenuazonic acid (TeA) Virulence factor
1. Introduction Alternaria alternata, one of the most cosmopolitan saprophytic fungal pathogens, also can infect numerous crops, causing yield and economic losses by at least 20% (Shafique et al., 2012). The mechanism of infection of A. alternata and the signaling pathways involved have been widely addressed (Chung, 2013; Akimitsu et al., 2014). Pathogenesis of A. alternata begins with host infection thus mutual recognition between host and pathogen is very important. Lectin, cutin, mannose glycoprotein, galactose and trehalose all can serve as signaling molecules that can be recognized by the host (Miyakawa et al., 2014; Poueymiro et al., 2014). Reactive oxygen species (ROS) produced by the host plant in the initial stage of infection can act as second messengers in activating an immune response (Heller and Tudzynski, 2011; Tudzynski et al., 2012).
* Corresponding author. E-mail address: [email protected] (S. Qiang). http://dx.doi.org/10.1016/j.plaphy.2017.03.002 0981-9428/© 2017 Elsevier Masson SAS. All rights reserved.
Consequently, the pathogen needs an effective antioxidant protection system to resist ROS from the host (Egan and Talbot, 2008; Samalova et al., 2014). ROS also regulate pathogenic fungal growth and differentiation; their balance is important during spore germination, appressorium development and hyphal tip growth (Egan et al., 2007; Guo et al., 2011; Kim et al., 2009). The response to ROS signal is regulated by the MAPK signaling pathway and/or some other pathways, which in concert also regulate the activity of peroxidases. In the tangerine pathotype of A. alternata, the Fus3/ Kss1 MAPK signaling pathway can singly or cooperatively regulate the pathogenicity with the AaAP1 gene (homologous transcription factor of YAP gene in Saccharomyces cerevisiae) that controls ROS levels in the pathogens (Lin et al., 2010). Thus ROS are key signaling molecules in pathogenicity. Upon infection, the pathogen forms appressoria that use turgor pressure generated by glycerol to penetrate the plant's epidermis (Howard et al., 1991). The pathogen destroys the host's cell wall by secreting cell wall degrading enzymes (Cho, 2015). Mycotoxins are common virulence factors in the host infection
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by plant pathogens, determining the interaction between the two organisms (Howlett, 2006). Most of the pathogens use toxins to kill plant cells obtaining nutrients from decaying tissue. Toxins could bind different targets, inhibit the activity of host plant enzymes and affect the biosynthesis of key metabolites. Some pathogens produce phytohormones that interfere with plant normal physiological processes and induce a ROS burst that damages host cells and tis€bius and Hertweck, 2009). Different toxins sues (Howlett, 2006; Mo have different modes of action. Toxins usually require photosensitizers to damage the host plant (Daub et al., 2005), bind target proteins (Chen et al., 2007, 2010), destroy biomembrane integrity (Abbas et al., 1994), deplete metal ions (Haas et al., 2008), block cell € bius and energy transfer (Meiss et al., 2008), induce apoptosis (Mo Hertweck, 2009; Stone et al., 2000), destroy the cytoskeleton € bius and Hertweck, 2009), and change gene expression (Mo € bius and Hertweck, 2009) to induce cell death. A remarkable (Mo non-host specific phytotoxin produced by A. alternata causing the brown leaf spot disease of croftonweed (Ageratina adenophora) is tenuazonic acid (TeA) (Qiang et al., 2010). TeA is a natural photosystem II inhibitor that blocks electron transport beyond QA by competing with QB for the QB-site in the D1 protein (Chen et al., 2007, 2010). TeA also inhibits the activity of chloroplast ATPase and Ferredoxin-NADP (þ) reductase (FNR). Oxidative damage resulting from TeA-induced ROS in chloroplasts of mesophyll cells is responsible for the TeA-triggered cell destruction and leaf necrosis (Chen et al., 2010). TeA besides damaging the host plant, may also effect A. alternata's pathogenicity by regulating ROS balance. But the mechanism by which TeA determines the infection of the host by A. alternata remains unclear. We here report studies on the mechanism of action of TeA in relation to pathogenicity using a toxin-deficient mutant strain.
2. Materials and methods 2.1. Biological materials and growth conditions A. alternata strain NEW was used as the reference, wild type; a toxin-deficient mutant strain, DHP001 obtained by restriction enzyme-mediated integration (REMI) is referred herein as the HP001 mutant strain. Both NEW and its derivative mutant were cultured on PSA medium (potato 200 g/L, sucrose 20 g/L, agar 15 g/ L) at 28 C. A. adenophora plants from seed were grown in the greenhouse (20e28 C, 70% humidity, 16/8 h). All foliage samples used in this study came from the upper second and third leaves of plants at the ten-leaf growth stage. Detached leaves were washed with ddH2O, placed on moistened filter paper and their petioles bundled with moistened cotton.
2.2. Monitoring the injury of A. adenophora leaves by A. alternata mycelia using Imaging-PAM Fungal mycelia were harvested from PS medium (potato 200 g/ L, sucrose 20 g/L) at 28 C, 120 rpm for 4e5 days. 0.1 g mycelia dried by filter paper were taken and mixed with 500 ml filtrates, then smashed using a homogenizer. An Imaging-PAM Chlorophyll Fluorometer (M Series, Heinz Walz GmbH, Germany) was used to measure photosynthesis after inoculating fungal mycelia onto A. adenophora leaves. The following recommended settings were applied in the study: pulse modulated measuring light intensity (ML) ¼ 2; measuring light pulse frequency (MF) ¼ 1; Gain ¼ 2; Damping ¼ 2; actinic light (AL) ¼ 8; and saturating flashes ¼ 10. The photosynthesis indices determined are presented in Table 1. 2.3. Effect of pathogen's filtrates on the pathogenicity of A. alternata mycelia A. adenophora leaves were inoculated with mycelium of the wild type and HP001 mutant alone and in combination with filtrates of each other. Photosynthetic indices were determined by ImagingPAM at 2, 6, 10, 14, and 18 h after inoculation and compared to those obtained from leaves inoculated with the wild type filtrate as control. The experiment was replicated three times. 2.4. Effect of TeA to the pathogenicity of A. alternata mycelium Wild type and HP001 mutant mycelium alone and in combination with 1000 ppm or 100 ppm TeA were inoculated onto A. adenophora leaves. The toxin applied alone at both concentrations was used as a control treatment. Photosynthetic indices were measured by Imaging-PAM as before. Also, HP001 mutant mycelium in combination with 0, 50, 60, 70, 80, 90 ppm TeA were inoculated onto A. adenophora leaves to determine if the recovery of pathogenicity of the HP001 mutant is TeA-concentration dependent. The experiment was replicated three times. 2.5. Effect of HP001 protein to the pathogenicity of A. alternata Wild type and HP001 mutant mycelia, HP001 mutant mycelium plus HP001 protein, and HP001 mutant mycelium plus vector pET28a were inoculated onto A. adenophora leaves and photosynthetic indices determined as before. 2.6. Microscopic observation of mycelium Mycelia of both the wild type and HP001 mutant and of HP001 mutant growing in presence of TeA were harvested from PS medium at 28 C, 120 rpm for 3 days and immobilized by
Table 1 Photosynthesis indices used in this study. Indexes Fo Fm Fv/Fm F Fm’ NPQ qN qP qL
Formulae
(Fm-Fo)/Fm
(Fm-Fm’)/Fm’ (Fm-Fm’)/(Fm-Fo’) (Fm’-F)/(Fm’-Fo’) (Fm’-F)/(Fm’-Fo’) Fo’/F ¼ qP Fo’/F
Descriptors Base fluorescence Maximum fluorescence after dark adaptation Maximal photochemical efficiency of PSII Current fluorescence yield in the presence of actinic illumination Maximal fluorescence yield Non-photochemical quenching Total non-photochemical quenching Photochemical quenching Photochemical quenching (Kramer Lake Model)
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glutaraldehyde to observe their cell wall characteristics by transmission electron microscopy (Hitachi H-7560). Fungal mycelia were also stained with aniline blue after inoculation onto A. adenophora leaves to observe the evolution of infection in the light microscope. The experiment was replicated three times. Mycelia were also inoculated onto detached onion epidermis, instead of A. adenophora epidermis that is difficult to remove from the leaves, for 24 h to observe and photograph the appressorium formation in light microscope. 2.7. ROS detection Intracellular ROS levels were monitored in mycelia using nitroblue tetrazolium (NBT) as described by Guo et al. (2011). Superoxide production was observed in light microscope and measured the gray scale of the light micrographs. The experiment was replicated three times. 2.8. Effect of epidermis removal on the pathogenicity of A. alternata The epidermis of A. adenophora leaves was manually removed using tweezers before inoculating HP001 mutant mycelium onto the subjacent leaf tissue for 24 h to observe its pathogenicity and detect the ROS content. The experiment was replicated three times. 2.9. Peroxidase enzyme activity Mycelia were ground under liquid nitrogen and suspended in PBS buffer as crude enzyme suspension. Laccase and ascorbate peroxidase activities were measured as described by Shelp et al. (1999) and Nakano and Asada (1981), respectively. Total glutathione peroxidase activity was measured according to a commercial protocol (Beyotime Biotechnology, S0058) and activity of ligninase was measured according to Faison and Kirk (1985). These tests were done at least three times, each with three replications. 2.10. Fus3 gene expression analyses Total RNA was extracted from mycelia growing in PS medium (28 C, 120 rpm for 1e7 days) and at 4-h intervals from previously inoculated A. adenophora leaves for 2e18 h to determine the expression levels of fus3. Changes during fungal development and infectious growth were calculated as 2-△△Ct, where △△Ct ¼ (Ct, target gene - Ct, actin gene)test condition - (Ct, WT - Ct, actin gene). qRT-PCR was performed with three independent pools of tissues in three sets of experimental replicates using the following primer pairs: actin_F: 50 -ggcaacattgtcatggtatg-3’; actin_R: 50 -gaggaagcaagaatggaac-3’; fus3_F: 50 -taccgagcacatcgagaa-3’; fus3_R: 50 -tcccaggaaggactacc0 3 3. Results 3.1. Photosynthetic system of the host plant was damaged before infection by A. alternata
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not penetrated the foliar tissue (Fig.1ABCD). Ten hours after inoculation, the foliar region inoculated with the wild type had begun developing lesions (Fig. 1D) and qL and qP and NPQ further decreased (Fig. 1A); the hyphae of the wild type were already colonizing the leaves and obvious disease spots developed by 14 h after inoculation (Fig. 1D). At this time, both the photochemical and non-photochemical quenching coefficients decreased drastically; quantum yield Y (II) of PSII also decreased (Fig. 1A). At 18 h after inoculation, disease spots continued to expand and the photosystem II reaction center became seriously damaged. In contrast, the regions inoculated with mutant still lacked disease spots and the chlorophyll fluorescence indexes remained at almost normal levels after 18 h (Fig. 1ABCD). The chlorophyll fluorescence indexes changed earlier than disease spots appeared indicating that active substances produced by hyphae could react early and cause photosynthetic system damage before the hyphae actually infected the host. Further experiments were conducted to determine if indeed the TeA toxin was involved in the onset of tissue damage and pathogen infection. 3.2. TeA toxin is essential for aerial hyphal growth The wild type fungus formed compact mycelium masses in liquid medium, in contrast to the sparse ones formed by the mutant. However, addition of TeA to the liquid PS medium restored the morphology of the mutant mycelia to closely resemble that of the wild type (Fig. 2A). Therefore TeA apparently contributes to maintaining mycelium morphology. 3.3. TeA toxin restores cell wall thickness Transmission electron micrographs revealed that the cell wall of the mutant was significantly thinner (0.07 mm) than that of the wild type (0.3 mm). By adding TeA, the cell wall of the mutant could be restored to half of the normal thickness (Fig. 2B and C). 3.4. Filtrates of cultured wild type mycelium partially restore the pathogenicity of the mutant The chlorophyll fluorescence index of leaf areas inoculated only with mutant hyphae, wild type culture filtrates, or wild type culture filtrates previously sterilized at high temperature had no significant variation 18 h after inoculation (Fig. 3). However, the chlorophyll fluorescence indexes of leaf sections inoculated with the mutant mycelium supplemented with a wild type culture filtrate, were similar to those in foliar areas inoculated by the wild type, regardless of high-temperature sterilization of the filtrate. Compared with 2 h after inoculation, these inoculated areas had significantly increased Y(NO), and significantly decreased NPQ and qN, at 18 h after inoculation (Fig. 3). The active ingredient in wild type culture filtrates, likely to be TeA, could make the mutant exhibit similar pathogenicity to that of the wild type, and the active ingredient is unlikely to be a large molecule such as a protein since it was not labile at high-temperature (data not show). 3.5. TeA toxin partially restores the pathogenicity of the mutant
Damage to the photosynthetic system of A. adenophora leaves inoculated with the wild type and mutant strains was observed by Imaging-PAM. Two hours after inoculation with either strain, no disease symptoms were observed in the inoculated regions, which also had normal chlorophyll fluorescence indexes (Fig.1ABC). Six hours after inoculation, disease spots were still absent, but the photochemical quenching coefficient (qL and qP) and the nonphotochemical quenching (NPQ) of the region inoculated with the wild type mycelium had decreased although its hyphae still had
The chlorophyll fluorescence indexes of leaf areas inoculated with the wild type were similar to those measured when the foliage was inoculated with mutant supplemented with the TeA toxin at either 1000 ppm or 100 ppm, whereas the Y(NO) significantly increased, and the NPQ and qN significantly decreased at 18 h after inoculation compared with at 2 h. This is a clear indication that the PSII reaction center of the plant was blocked and that photodynamic damage occurred because of the inability of the
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Fig. 1. Fluorescence image of Alternaria alternata hyphae at the infection stage. A. The chlorophyll fluorescence indexes of Ageratina adenophora leaves from time of hyphal inoculation; B. Light photographs and C. Fluorescence images (Fv/Fm) of A. adenophora leaves inoculated by wild type (left circle) and HP001 mutant (△HP001, right circle) mycelia showing the development of lesions; D. Light micrographs of aniline-blue stained wild type mycelia growing in A. adenophora leaves. Bars equal 30 mm.
photosynthetic apparatus to dissipate excess light energy. The chlorophyll fluorescence indexes in the areas of leaves inoculated with the mutant or treated only with either 1000 ppm or 100 ppm TeA toxin did not change during the observation period, i.e. from two to 18 h after inoculation (Fig. 4) (The results of 1000 ppm TeA toxin treatment were confirmed in a separate bioassay in Fig. S1). Hyphae of the mutant mixed with TeA toxin at concentrations ranging from 50 to 100 ppm were inoculated on leaves to study the concentration-dependent effect of the toxin on pathogenicity. Damage to the photosynthetic apparatus was detected at 24 h after inoculation only after the addition of the highest concentration of 100 ppm (Fig. S2). Moreover, the addition of HP001 expression product had no effect on pathogenicity (Fig. S3). These results further point to TeA toxin likely being the biologically active ingredient in wild type culture filtrates, thus suggesting an important role of the toxin on pathogenicity.
3.6. TeA toxin promotes appressorium formation Hyphae of the wild type and mutant inoculated on onion epidermis grew normally, but those of the mutant failed to form appressoria (Fig. 5A and B). The wild type hyphae were difficult to remove manually from the onion epidermis at 24 h after inoculation but the mutant hyphae easily detached at this time because they lacked appressoria to attach them to the epidermis. However, mutant hyphae formed some appressoria structures when TeA toxin was added (Fig. 5C and D). This might be a reason for the
mutant partial recovery of pathogenicity by addition of TeA toxin.
3.7. TeA reduces the ROS content of the HP001 mutant The hyphal tip of the mutant contained about four times more ROS than that of the wild type, regardless if cultured on PSA medium or on a wet slide surface (Fig. S4). When infecting onion epidermis and A. adenophora leaves, the mutant contained seven to five times more ROS that the wild strain (Figs. 6 and 7A). The ROS concentration of mutant was reduced to wild-type levels during the infection process after adding 100 ppm TeA (Figs. 6A and 7A); the lowest concentration tested (50 ppm TeA) was unable to elicit this effect (Fig. 7A).
3.8. TeA toxin increases peroxidase activity of the HP001 mutant The peroxidase activity, including laccase, total glutathione peroxidase, ligninase, and ascorbate peroxidase, of both the wild type and mutant increased throughout the experimental period. But the peroxidase activity of the wild type was significantly higher than that of the mutant (Fig. 8ABCD). Adding TeA significantly enhanced the activity of laccase, ascorbate peroxidase, and total glutathione peroxidase of the mutant during the hyphal growing period, but not to the same level found in the wild type (Fig. 8ABC). The ligninase activity of the mutant was not affected by the addition of TeA (Fig. 8D).
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Fig. 2. Phenotype of growing mycelia and cell wall of Alternaria alternata. A. Phenotype of mycelia growing in liquid medium; B. Transmission electron micrographs of cross section of mycelium showing the cell wall. Bars equal 1 mm; C. Thickness of cell wall. Each column represents the mean ± the standard error from three independent experiments, each with at least three replicates. Letters above error bars indicate significance at P < 0.05.
Fig. 3. Contrast analyses of chlorophyll fluorescence parameters of wild type culture filtrate interacting with the HP001 mutant. 1. Wild type hyphae; 2. Mutant hyphae; 3. Mutant hyphae þ wild type culture filtrate (without autoclaving); 4. Mutant hyphae þ wild type culture filtrate (autoclaved); 5. Wild type culture filtrate (not-autoclaved); 6. Wild type culture filtrate (autoclaved). Each column represents the mean value ± the standard error from three independent experiments, each with at least three replicates. Columns labeled with the same letter are not significantly different (P < 0.05). Fluorescence images are color coded according to the patterns shown next to the images. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Contrast analyses of chlorophyll fluorescence parameters of TeA interacting with the HP001 mutant. 1. Wild type hyphae; 2. Mutant hyphae; 3. Mutant hyphae þ1000 ppm TeA; 4. Mutant hyphae þ100 ppm TeA; 5.1000 ppm TeA; 6.100 ppm TeA. Each column represents the mean value ± the standard error from three independent experiments, each with at least three replicates. Columns labeled with the same letter are not significantly different (P < 0.05). Fluorescence images are color coded according to the patterns shown next to the images. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Light micrographs of Alternaria alternata wild type and HP001 mutant hyphae infecting the epidermis of onion. A., B. Wild type and mutant hyphae infecting the epidermis of onion, respectively; C, D. Infection of leaf epidermis by mutant hyphae to which TeA toxin was added. Arrows point to appressoria. Bars equal 50 mm.
3.9. Leaves without their epidermis reduce the ROS content of HP001 mutant When the mutant mycelia were inoculated onto host leaves areas without epidermis, the ROS of mutant was reduced to about a half (Figs. S5A and B). This further suggests the involvement of TeA
in the fungus recognition of the host epidermis. 3.10. HP001 mutant infects host leaves whose epidermis has been removed The mutant caused disease when it was inoculated on leaves
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Fig. 6. Detection in light micrographs of ROS in the tip of hyphae of wild type and HP001 mutant (△HP001) under the epidermis of onion. A. Light micrographs of the superoxide in wild type and HP001 mutant under epidermis of onion. Bars equal 50 mm; B. Measurement of ROS content by the gray scale of the photomicrographs. Each column represents the mean ± the standard error from three independent experiments, each with at least three replicates. Columns labeled with the same letter are not significantly different (P < 0.05).
Fig. 7. Detection in light micrographs of ROS and illustration of lesions on Ageratina adenophora after addition of TeA at different concentrations. A. Photomicrographs of superoxide in photomicrographs of wild type, HP001 mutant (△HP001) and HP001 mutant under two concentrations of TeA. Bars equal 10 mm; B. Measurement of ROS content by the gray scale of the photographs. Each column represents the mean ± the standard error from three independent experiments, each with at least three replicates. Columns labeled with the same letter are not significantly different (P < 0.05). C. Lesions caused by HP001 mutant in combination with TeA at two concentrations of TeA compared with the wild type and TeA by itself (according to labels above circles).
whose epidermis had been previously removed (Fig. S5C). Evidently the epidermis is a major barrier for mutant infection but not for the wild type. This indicates that TeA may play a role in recognizing the host and breaking through the epidermis barrier. 3.11. TeA does not affect the Fus3/Kss1 MAPK signaling pathway Based on qRT-PCR, the wild type had significantly higher expression of the fus3 gene than the mutant, regardless of the stage of hyphal growth or hyphal infection of A. adenophora leaves. At the hyphal growth stage, the gene expression level peaked on the sixth day (Fig. 9A) and during the hyphal infection stage, the expression level reached peak at the 14th h (Fig. 9B). However, the expression level of the gene was not substantially improved by adding TeA to
the mutant. 4. Discussion Our lab has systematically evaluated the herbicidal activity of TeA to assess its potential as a bio-herbicide (Qiang et al., 2010). TeA was found to be a novel photosystem II inhibitor (Chen et al., 2007, 2010) whose phytotoxic action is mediated by the generation of singlet oxygen (Chen et al., 2015). In this study, we established the mechanism by which TeA determines the infection of the host by A. alternata and its role as a key virulence factor of the pathogen. The first step of the infection process is the mutual recognition of host plant and pathogen, which could regulate mycelial growth and appressorium formation (Aguirre et al., 2005; Lalucque and
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Fig. 8. Determination of peroxidase activity in wild type, HP001 mutant (△HP001) and the HP001 mutant after addition of TeA (DHP001 þ TeA) during a six-day growing period of Alternaria alternata. A. Laccase; B. Ascorbate peroxidase; C. Glutathione peroxidase; D. Ligninase. Each column represents the mean value ± the standard error from three independent experiments, each with at least three replicates. Columns labeled with the same letter are not significantly different (P < 0.05).
Fig. 9. Quantitative gene-expression analysis of fus3 gene among the wild type strain (WT), the HP001 mutant (DHP001), and the HP001 mutant after addition of TeA (DHP001 þ TeA) at the growing (A) and infection stages (B) of Alternaria alternata. Error bars represent the standard deviations.
Silar, 2003). ROS can function as molecular signals to regulate fungal sexual reproduction, growth, development, infection, and pathogenicity (Egan et al., 2007; Sun et al., 2006). Thus the ROS balance is a determinant of pathogenicity (Aguirre et al., 2005; Egan et al., 2007; Guo et al., 2011; Kim et al., 2009; Tanaka et al., 2006), and a complete peroxidase system in the fungus degrading excessive ROS would relieve damage to its cellular structure (Foley et al., 2016). An excessive accumulation of ROS in the conidia of A. brassicicola breaks the intracellular redox dynamic balance, ultimately
resulting in loss of pathogenicity (Kim et al., 2009). Results of this study show that the HP001 mutant recovers its colony morphology, reduces the ROS content in mycelia, increases the activity of peroxidase and recovers the pathogenicity after addition of exogenous TeA toxin. According to our results the apparent reason for the loss of pathogenicity in the mutant is the breaking of the ROS balance. Previous studies showed that a burst of ROS could be used as a signal during pathogen infection (Egan et al., 2007). But the content of ROS in the mutant is much more than that needed during the interaction between pathogen and host plant, with the
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disruption of ROS homeostasis being responsible for the loss of pathogenicity. Exogenous betaine and CaCl2 increase the activity of antioxidant enzymes that scavenge excessive ROS in Cystofilobasidium infirmominiatum and Debaryomyces hansenii, which could be conducive to improve the vitality of the fungi (An et al., 2012; Liu et al., 2011). Similarly, our research supports the notion that TeA could improve the peroxidase activity of the mutant and the scavenging of excessive ROS that would allow it recovering the ability to respond to the exogenous signals and recover its pathogenicity. Infection structure development is the key step in the colonization of the host plant by A. alternata after signal recognition. The formation of the appressorium is related to the molecular recognition interaction between the pathogen and its host (Deising et al., 2000; Ryder and Talbot, 2015). In M. oryzae, the formation of the appressorium is mainly related to the three signal transduction pathways, cAMP signaling pathway, MAPK signaling pathway and Ca2þ signaling pathway (Dean, 1997; Xu, 2000). Among them, the PMK1 signaling pathway (homologous with the yeast Fus3/Kss1 signaling pathway) is closely related to the formation and maturation of the appressorium. PMK1 was highly expressed during the formation of the appressorium, and a PMK1 mutant was unable to form it (Xu, 2000). Mst50, mst11, and mst7 located upstream of the PMK1 pathway all play an important role in the formation of the appressorium (Zhang et al., 2016; Zhao et al., 2005). The signaling pathway and some cooperating pathways regulate the response of fungi to ROS, and also the activity of a series of peroxidases in fungi (Lin et al., 2010). In our study, the mutant hyphae grew normally on the surface of host leaves, but could not infect them, mainly because its failure to form the appressorium. Excessive ROS in mycelial cell of the HP001 mutant may hinder the HP001 mutant to actuate the signaling pathway for the formation of the appressorium through the molecular recognition interaction between the pathogen and its host which was mediated by ROS. The mutant could recover the ability to form appressorium, infect host leaves and damage them after adding exogenous TeA. TeA could recover the mutant's responses to the exogenous ROS-mediated signals for the formation of the appressorium through improving the peroxidase activity and the scavenging of excessive ROS. This suggested that TeA toxin may be directly involved in the formation of appressorium during the infection process. Although the mutant lost its ability to infect the host, its hyphae could infect and colonize host leaf areas devoid of epidermis and thus it exhibited some pathogenicity. We posit that TeA toxin may play an important role in the hyphae entering into the mesophyll cell successfully at a primary infection stage. Last, the hyphae complete the infection stage and produce the characteristic disease spots. Being generally saprophytic, the fungi could kill host cells using toxins, obtain nutrients from the host plant and cause disease spots. In this research, the photosynthetic apparatus began degrading when wild type hyphae were inoculated on host leaves for 6 h, and excess light had damaged the photosynthetic apparatus. At the same time, hyphae did not infect the host 6 h after inoculation, thus disease spots maybe caused by some secondary metabolites. As it is difficult to detect these secondary metabolites, it was not possible to determine which one could cause damage. The ability of the mutant to infect host leaves devoid of epidermis clearly shows that the epidermis is a major barrier for the mutant that otherwise could infect its host and cause disease provided that the hyphae enter the mesophyll cells. We speculate that the restoration of pathogenicity of the mutant by the addition of TeA is an indication that this toxin begins damaging PSII before hyphae actually infects the host. Lesions would then be the result of both the action of the toxin and hyphae. Furthermore, after successful colonization of the host, TeA toxin may also help the
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pathogen in killing host cells, whose contents serve as nutrients to the pathogen thus facilitating the expansion of infection. It remains to verify this experimentally. The Fus3/Kss1 MAPK signaling pathway is the most widely studied MAPK signaling pathway in phytopathogenic fungi. Several Fus3/Kss1 homologous genes have been isolated that are also involved in sporulation, vegetative growth, resistance to oxidative stress, and melanin metabolism (Xu, 2000). Although the function of the Fus3/Kss1 MAPK signaling pathway is conserved, its role in several fungi is slightly different. In Alternaria spp., the Aafus3 gene has many functions, including in spore development, fungal infection, melanin biosynthesis, and pathogenicity (Lin et al., 2010). To further explore the molecular mechanism of the partial recovery of pathogenicity after adding TeA, we determined the expression of fus3 genes in the fungus’ signaling pathway. The expression of the fus3 gene was not significantly increased after adding TeA to the mutant. Since addition of the TeA toxin partially restored the cell wall thickness, appressorium formation and the pathogenicity of the HP001 mutant, we surmise that the metabolic pathway of TeA toxin that is involved in pathogenicity may be located downstream of the MAPK signaling pathway. It may regulate physiological processes like ROS metabolism, as well as appressorium and cell wall formation and pathogenicity, combined with MAPK signaling pathway, but the specific signaling networks need to be further studied. We can summarize the infection process of A. alternata. The phytopathogenic fungus starts secreting TeA toxin before infecting its host. The TeA toxin inhibits host photosynthetic electron transport in PSII leading to a ROS burst and damage of the epidermal and mesophyll cells. The hyphae forms appressoria after identifying the host epidermis then breaks this epidermal barrier, infecting and colonizing the host tissues to complete the infection process. The mutant produces insufficient TeA to damage the epidermis of the host, and has a high ROS content in the hypha cell, which causes the pathogen to be unable to respond to the host signal, failing to form the appressorium required to infect the host, and thereby losing pathogenicity. Thus TeA toxin is the key virulence factor for A. alternata infection of its host. Contributions Sheng Qiang (SQ) designed the study. Ye Kang (YK), Hongwei Feng (HF), Jingxu Zhang (JZ) and Shiguo Chen (SC) performed synthesis experiments and characterization. YK, JZ and SQ interpreted the data and drafted the manuscript. SQ, YK and Bernal E. Valverde (BV) majorly revised the manuscript. All authors contributed to and approved the final manuscript. Acknowledgements This work was supported by National Technology Research and Development Program of China and Science and Technology Pillar Program of Jiangsu Province [BE2014397]. The TeA toxin was kindly provided by Professor Chunlong Yang from the College of Sciences of Nanjing Agricultural University. The authors also gratefully thank Min Zhang, Qin Yao and Tingting Ran from the College of Life Science of Nanjing Agricultural University for their help to the experiments. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2017.03.002.
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References Abbas, H.K., Tanaka, T., Duke, S.O., Porter, J.K., Wray, E.M., Hodges, L., Sessions, A.E., Wang, E., Merrill Jr., A.H., Riley, R.T., 1994. Fumonisin-and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases. Plant Physiol. 106, 1085e1093. Aguirre, J., Ríos-Momberg, M., Hewitt, D., Hansberg, W., 2005. Reactive oxygen species and development in microbial eukaryotes. Trends Microbiol. 13, 111e118. Akimitsu, K., Tsuge, T., Kodama, M., Yamamoto, M., Otani, H., 2014. Alternaria hostselective toxins: determinant factors of plant disease. J. Gen. Plant Pathol. 80, 109e122. An, B., Li, B., Qin, G., Tian, S., 2012. Exogenous calcium improves viability of biocontrol yeasts under heat stress by reducing ROS accumulation and oxidative damage of cellular protein. Curr. Microbiol. 65, 122e127. Chen, S., Kim, C., Lee, J.M., Lee, H.A., Fei, Z., Wang, L., Apel, K., 2015. Blocking the QBbinding site of photosystem II by tenuazonic acid, a non-host-specific toxin of Alternaria alternata, activates singlet oxygen-mediated and EXECUTERdependent signalling in Arabidopsis. Plant Cell Environ. 38, 1069e1080. Chen, S., Xu, X., Dai, X., Yang, C., Qiang, S., 2007. Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in QB-site of Chlamydomonas reinhardtii. BBA-Bioenerg. 1767, 306e318. Chen, S., Yin, C., Qiang, S., Zhou, F., Dai, X., 2010. Chloroplastic oxidative burst induced by tenuazonic acid, a natural photosynthesis inhibitor, triggers cell necrosis in Eupatorium adenophorum Spreng. BBA-Bioenerg. 1797, 391e405. Cho, Y., 2015. How the necrotrophic fungus Alternaria brassicicola kills plant cells remains an enigma. Eukaryot. Cell 14, 335e344. Chung, K.R., 2013. Mitogen-activated protein kinase signaling pathways of the tangerine pathotype of Alternaria alternata. MAP Kinase 2, 4. Daub, M.E., Herrero, S., Chung, K.R., 2005. Photoactivated perylenequinone toxins in fungal pathogenesis of plants. FEMS Microbiol. Lett. 252, 197e206. Dean, R.A., 1997. Signal pathways and appressorium morphogenesis. Annu. Rev. Phytopathol 35, 211e234. Deising, H.B., Werner, S., Wernitz, M., 2000. The role of fungal appressoriain plant infection. Microbes Infect. 2, 1631e1641. Egan, M.J., Talbot, N.J., 2008. Genomes, free radicals and plant cell invasion: recent developments in plant pathogenic fungi. Curr. Opin. Plant Boil 11, 367e372. Egan, M.J., Wang, Z.Y., Jones, M.A., Smirnoff, N., Talbot, N.J., 2007. Generation of reactive oxygen species by fungal NADPH oxidases is required for rice blast disease. P. Natl. Acad. Sci. U. S. A. 104, 11772e11777. Faison, B.D., Kirk, T.K., 1985. Factors involved in the regulation of a ligninase activity in Phanerochaete chrysosporium. Appl. Environ. Microb. 49, 299e304. Foley, R.C., Kidd, B.N., Hane, J.K., Anderson, J.P., Singh, K.B., 2016. Reactive oxygen species play a role in the infection of the necrotrophic fungi, Rhizoctonia solani in wheat. PLoS One 11, e0152548. Guo, M., Chen, Y., Du, Y., Dong, Y., Guo, W., Zhai, S., Zhang, H., Dong, S., Zhang, Z., Wang, Y., Wang, P., Zheng, X., 2011. The bZIP transcription factor MoAP1 mediates the oxidative stress response and is critical for pathogenicity of the rice blast fungus Magnaporthe oryzae. PLoS Pathog. 7, e1001302. Haas, H., Eisendle, M., Turgeon, B.G., 2008. Siderophores in fungal physiology and virulence. Annu. Rev. Phytopathol. 46, 149e187. Heller, J., Tudzynski, P., 2011. Reactive oxygen species in phytopathogenic fungi: signaling, development, and disease. Annu. Rev. Phytopathol. 49, 369e390. Howard, R.J., Ferrari, M.A., Roach, D.H., Money, N.P., 1991. Penetration of hard substrates by a fungus employing enormous turgor pressures. P. Natl. Acad. Sci. U. S. A. 88, 11281e11284. Howlett, B.J., 2006. Secondary metabolite toxins and nutrition of plant pathogenic fungi. Curr. Opin. Plant Biol. 9, 371e375. Kim, K.H., Willger, S.D., Park, S.W., Puttikamonkul, S., Grahl, N., Cho, Y.,
Mukhopadhyay, B., Cramer, R., Lawrence, C.B., 2009. TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen. PLoS Pathog. 5, e1000653. Lalucque, H., Silar, P., 2003. NADPH oxidase: an enzyme for multicellularity? Trends Microbiol. 11, 9e12. Lin, C.H., Yang, S.L., Wang, N.Y., Chung, K.R., 2010. The FUS3 MAPK signaling pathway of the citrus pathogen Alternaria alternata functions independently or cooperatively with the fungal redox-responsive AP1 regulator for diverse developmental, physiological and pathogenic processes. Fungal Genet. Biol. 47, 381e391. Liu, J., Wisniewski, M., Droby, S., Vero, S., Tian, S., Hershkovitz, V., 2011. Glycine betaine improves oxidative stress tolerance and biocontrol efficacy of the antagonistic yeast Cystofilobasidium infirmominiatum. Int. J. Food Microbiol. 146, 76e83. Meiss, E., Konno, H., Groth, G., Hisabori, T., 2008. Molecular processes of inhibition and stimulation of ATP synthase caused by the phytotoxin tentoxin. J. Biol. Chem. 283, 24594e24599. Miyakawa, T., Hatano, K., Miyauchi, Y., Suwa, Y.I., Sawano, Y., Tanokura, M., 2014. A secreted protein with plant-specific cysteine-rich motif functions as a mannose-binding lectin that exhibits antifungal activity. Plant Physiol. 166, 766e778. €bius, N., Hertweck, C., 2009. Fungal phytotoxins as mediators of virulence. Curr. Mo Opin. Plant Biol. 12, 390e398. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867e880. , A.C., François, J.M., Parrou, J.L., Peeters, N., Genin, S.A., 2014. Poueymiro, M., Cazale Ralstonia solanacearum Type III effector directs the production of the plant signal metabolite trehalose-6-phosphate. mBio 5 e02065e14. Qiang, S., Wang, L., Wei, R., Zhou, B., Chen, S., Zhu, Y., Dong, Y., An, C., 2010. Bioassay of the herbicidal activity of AAC-toxin produced by Alternaria alternata isolated from Ageratina adenophora. Weed Technol. 24, 197e201. Ryder, L.S., Talbot, N.J., 2015. Regulation of appressorium development in pathogenic fungi. Curr. Opin. Plant Biol. 26, 8e13. Samalova, M., Meyer, A.J., Gurr, S.J., Fricker, M.D., 2014. Robust anti-oxidant defences in the rice blast fungus Magnaporthe oryzae confer tolerance to the host oxidative burst. New Phytol. 201, 556e573. Shafique, S., Majeed, R.A., Shafique, S., 2012. Cymbopogon citrates: a remedy to control selected Alternaria species. J. Med. Plants Res. 6, 1879e1885. Shelp, B.J., Bown, A.W., McLean, M.D., 1999. Metabolism and functions of gammaaminobutyric acid. Trends Plant Sci. 4, 446e452. Stone, J.M., Heard, J.E., Asai, T., Ausubel, F.M., 2000. Simulation of fungal-mediated cell death by Fumonisin B1 and selection of Fumonisin B1-resistant (fbr) Arabidopsis mutants. Plant Cell 12, 1811e1822. Sun, C.B., Suresh, A., Deng, Y.Z., Naqvi, N.I., 2006. A multidrug resistance transporter in Magnaporthe is required for host penetration and for survival during oxidative stress. Plant Cell 18, 3686e3705. Tanaka, A., Christensen, M.J., Takemoto, D., Park, P., Scott, B., 2006. Reactive oxygen species play a role in regulating a fungus-perennial ryegrass mutualistic interaction. Plant Cell 18, 1052e1066. Tudzynski, P., Heller, J., Siegmund, U., 2012. Reactive oxygen species generation in fungal development and pathogenesis. Curr. Opin. Microbiol. 15, 653e659. Xu, J.R., 2000. MAP kinases in fungal pathogens. Fungal Genet. Biol. 31, 137e152. Zhang, S., Jiang, C., Zhang, Q., Qi, L., Li, C., Xu, J.R., 2016. Thioredoxins are involved in the activation of the PMK1 MAP kinase pathway during appressorium penetration and invasive growth in Magnaporthe oryzae. Environ. Microbiol. http:// dx.doi.org/10.1111/1462-2920.13315. Zhao, X., Kim, Y., Park, G., Xu, J.R., 2005. A mitogen-activated protein kinase cascade regulating infection-related morphogenesis in Magnaporthe grisea. Plant Cell 17, 1317e1329.