A Glycosylphosphatidylinositol-anchored protein from   Alternaria alternata  triggers cell death and negatively modulates immunity responses in chrysanthemum

doi:10.21203/rs.3.rs-4847532/v1

Download PDF

Research Article

A Glycosylphosphatidylinositol-anchored protein from Alternaria alternata triggers cell death and negatively modulates immunity responses in chrysanthemum

https://doi.org/10.21203/rs.3.rs-4847532/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Chrysanthemum black spot disease is a common fungal disease caused by A. alternata, which has severely hindered the development of the chrysanthemum industry. However, there are few reports on pathogenic factors in A. alternata, especially regarding GPI-APs. In this study, we identified a GPI-AP, Aa049, from A. alternata. Bioinformatics predictions suggest the presence of a GPI anchoring modification site at the C-terminus of its amino acid sequence, which is relatively conserved among different Alternaria Nees. Transient overexpression of Aa049 in Nicotiana benthamiana can induce plant cell death (PCD), and the appearance of necrosis depends on its native signal peptide. Compared with the wild-type strain, the morphology and growth rate of the colony and mycelia of the ΔAa049 deletion mutants did not change. Still the integrity of the cell wall was damaged, and the virulence of the strain was significantly reduced, indicating that Aa049 plays an essential role as a pathogenic factor in the infection process of A. alternata. Furthermore, the results of quantitative real-time PCR (qRT-PCR) and physiological indicators suggested that the virulence of Aa049 may be exerted through the synthesis and clearance pathways of ROS. This study reveals that GPI-APs in A. alternata can act as virulence factors to aid pathogen invasion, providing a potential target for the development of future biopesticides.

Alternaria alternata

Aa049

cell death

immunity

chrysanthemum

Glycosylphosphatidylinositol-anchored protein (GPI-AP) Aa049 works as a key pathogenic factor to assist A. alternata in infecting plants, which is associated with the reactive oxygen species (ROS) pathway

Alternaria Nees, a widely distributed fungus worldwide, has a broad host range and survives as an endophyte in many plants. Due to its adaptability to environmental stressors such as UV radiation, low temperatures, and water stress conditions, it also thrives as a saprophyte. As a primary pathogen of many economically important crops, Alternaria Nees severely affects yield and quality (Crudo, et al. 2019; DeMers 2022). Some races of Alternaria can also infect animals and humans, being implicated as major pathogens causing allergic respiratory diseases and phaeohyphomycosis (Fernandes, et al. 2023). Furthermore, its toxin production poses a severe threat to human health through food contamination. The economic losses caused by Alternaria-induced plant diseases in agricultural production are substantial, yet no highly effective fungicides have emerged. Therefore, in-depth and meticulous studies of the pathogenic mechanisms of Alternaria Nees are necessary to establish a solid theoretical foundation for designing pesticide targets and breeding resistant varieties in the future.

As a necrotrophic fungus, Alternaria spp. initiates complex and precise pathogenic processes when infecting plants, which can generally be divided into four steps: (1) adherence to the surface of the host ; (2) formation of infection structures; (3) invasion of the host; and (4) colonization and proliferation within the host (Li, et al. 2022). The pathogenic mechanisms of Alternaria fungi involve various factors, including mechanical penetration (Howard, et al. 1991); secretion of effector proteins such as AbrNLPs (Duhan, et al. 2021), AsCEPs (Wang, et al. 2023; Wang, et al. 2022), AsCFEMs (Qiu, et al. 2024); regulation by transcription factors like AbSte12 (Cho, et al. 2009), AbVf19 (Srivastava, et al. 2012), Amr1 (Cho, et al. 2012), AbPf2 (Cho, et al. 2013); cell wall-degrading enzymes (Li, et al. 2024) and metabolic products including toxins, melanin, non-ribosomal peptides, mannitol, etc. (Cho 2015). Additionally, MAPK signaling pathways such as AaSLT2 (Yago, et al. 2011) and AbSte7 (Lu, et al. 2019) are involved, but research on GPI-APs remains unexplored.

Glycosylphosphatidylinositol anchoring modification is a post-translational modification widely present in eukaryotes such as plants, animals, and fungi. This process occurs in the endoplasmic reticulum, and GPI-APs are then transported to the cell membrane or cell wall, where they exert biological functions (Fujita and Kinoshita 2012). Typically, GPI-APs consist of a signal peptide at the N-terminus, hydrophobic amino acids at the C-terminus, and the C-terminal GPI-anchored binding site ω, where ω is the key site for GPI modification (Nather and Munro 2008). GPI-APs in eukaryotes have diverse functions, including cell adhesion, signal transduction, enzyme catalysis, and cell wall integrity maintenance. Previous studies have highlighted the significant role of GPI-APs in the fungal infection of plants. Multiple GPI-APs are present in the wheat pathogen Fusarium graminearum, where GPI7 encodes a phosphoethanolamine transferase. Mutants of GPI7 exhibit slowed radial growth rate, malformed macroconidia, cell wall defects, and significantly reduced pathogenicity (Rittenour and Harris 2013). Similarly, GPI7 in Magnaporthe oryzae contributes significantly to pathogenicity by regulating the localization and abundance of cell wall Gel (Glucan elongation) family proteins, which are crucial for maintaining cell wall integrity (Liu, et al. 2020). In Ustilaginoidea virens, a Ser-Thr-rich Glycosyl-phosphatidyl-inositol-anchored protein (SGP1) acts as a Microbe-Associated Molecular Pattern (MAMP) triggering immune responses in rice leaves, while in spikelets it is essential for U. virens virulence (Song, et al. 2021). In Cryphonectria parasitica, the GPI-AP, CpGAP1, acts not only as a virulence factor in plants but also functions as a resistance factor against the fungal virus CHV1 (Chun, et al. 2022). SsGSR1 in Sclerotinia sclerotiorum encodes a glycine and serine-rich GPI-AP, which induces local cell death and Pattern-Triggered Immunity (PTI) responses in plants. Deletion mutants of SsGSR1 disrupt cell wall structure and integrity in S. sclerotiorum, significantly reducing its pathogenicity (Hu, et al. 2023). Despite advances in genome sequencing technology leading to the identification of GPI-APs in other eukaryotes, there have yet to be any reports on GPI-APs in Alternaria Nees to date.

Chrysanthemum is one of the world's four major cut flowers, valued for its uses in tea, medicine, and ornamental purposes, promising a strong market potential. A. alternata can infect various plants of the Asteraceae family, such as Aster, Cornflower, Cosmos, Rheuifolia, Gerbera, Purple-backed Geranium, Silver Ragwort, Cineraria, Sunflower and Marigold, causing black spot disease (Enjoji 1931; Pelser, et al. 2007; Takano 2001). Initially, small brown lesions develop on the leaves, which are circular or irregular in shape, progressing to perforated lesions in advanced stages. Under conditions of high temperature and humidity, the pathogen can rapidly spread throughout the field, significantly reducing the ornamental value and landscaping effects of Asteraceae plants, leading to irreversible economic losses. Currently, our understanding of the pathogenic factors of A. alternata during chrysanthemum infection is limited. Only one gene, AaBRE1, encoding an E3 ubiquitin ligase from A. alternata, has been identified, which mediates histone 2B (H2B) monoubiquitination and lysine 4 on histone 3 trimethylation (H3K4me3), regulating hyphal growth and pathogenicity (Liu, et al. 2020). In this study, we identified a GPI-AP in the pathogenic fungus A. alternata, whose overexpression in plants induces localized cell death. This protein plays a crucial role in the integrity of Alternaria's cell wall structure and virulence. Moreover, it may regulate plant resistance to A. alternata through the ROS pathway. These findings provide insights into understanding the mechanisms of plant-microbe interactions for better management strategies.

Plant materials and growth conditions

The chrysanthemum ‘Jinba’ and N. benthamiana used in this study were provided by the Chrysanthemum Genetics and Germplasm Innovation Team of Nanjing Agricultural University. All plants were planted in a mixed soil with a volume ratio of 1:3 of nutrient soil: vermiculite, a constant temperature of 22°C, a relative humidity of 70%, and a long-day greenhouse (16 h light, 8 h dark).

Gene cloning and sequence analysis

Partial Aa049 gene sequence was obtained from a transcriptome database, in which chrysanthemum leaves were infected with A. alternata (Liu, et al. 2020). Then the sequence was aligned in NCBI-BLAST to obtain full-length coding region sequence (CDS) fragments, and primers were designed (Table S1) for PCR amplification. The PCR product was connected to the pEASY-Blunt cloning vector (TransGen, Beijing, China) and transformed into Escherichia coli DH5α for sequencing. Then the amino acid sequences were obtained by DNAMAN software based on the CDS sequences. Further, the amino acid sequences were placed in SignalP6.0 (https://services.healthtech.dtu.dk/services/SignalP-6.0/), BIG-PI Fungal Predictor (https://mendel.imp.ac.at/gpi/fungi_server.html), and TMHMM 2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/) for protein information prediction. In addition, the top 20 most similar protein amino acid sequences obtained in NCBI-BLST were compared in DNAMAN and MEME (https://meme-suite.org/meme/tools/meme) to predict the conserved motifs. These sequences were input into clustalx1.81 software for alignment, and then the phylogenetic tree was constructed by MEGA5.1 software using the neighbor-joining method.

Inoculation treatment on chrysanthemum

We cultivated wild-type A. alternata F20 or ΔAa049 mutant strains on potato dextrose agar (PDA) medium for 5–7 days, then took a fungal cake with a diameter of about 4 mm from the edge of the medium and placed it in fresh potato dextrose water medium at 28°C and 200 rpm overnight. The next day, we took the mycelial balls shaken before, ground them thoroughly and transferred them to fresh potato dextrose water medium, and continued to culture at 28°C and 200 rpm for 1 day. When inoculating, we enriched one mycelial ball for every 1 ml of mycelium suspension. Chrysanthemum seedlings with 10–13 leaves of consistent growth were selected and inoculated with the mycelial balls on the back of the third fully expanded leaf, with two inoculation points on each leaf. The leaves inoculated with the pathogen were covered with a transparent plastic bag to keep them moist and placed at 28°C in the dark for 2 days, and then the diseased leaves were photographed.

qRT-PCR analysis

To analyze the expression pattern of Aa049 during the infection of A. alternata, we sampled the third fully expanded leaf (counted from the shoot apex) of chrysanthemum inoculated with A. alternata at different time points (0, 6, 24, 48, 72 hours post-inoculation), extracted RNA using quick RNA isolation kit (Huayueyang Biotechnology, Beijing, China), and further reverse transcribed into cDNA using Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver.2 (Accurate Biotechnology, Changsha, China). According to the instructions, we used 20 µL of SYBR Green Pro Taq HS (Accurate Biotechnology, Changsha, China) to perform qRT-PCR on a Roche LightCycler480 II instrument (Roche, Basel, Switzerland), using AaActin as the internal reference. The relative transcription level was calculated using the 2^−ΔΔCt method (Livak and Schmittgen, 2001). Three biological replicates were set for each sample, and each biological replicate had three technical replicates.

Yeast secretion and TTC (2, 3, 5-triphenyl tetrazolium chloride) assay

We used the ready-to-use seamless cloning kit (Sangon Biotech, Shanghai, China) to insert the full-length CDS of Aa049 (including the signal peptide) and the Aa049-SP gene fragment with only the signal peptide into the pSUC2 vector. The above vector plasmids were transformed into the yeast strain YTK12, incubated on CMD/-W medium (the YTK12 strain was streaked on the YPDA plate simultaneously) at 30°C for 3 days. In this experiment, we used pSUC2::Avr1b, which has been reported to have a secretory function, as a positive control, and pSUC2::Mg87, which has no secretory function, the empty vector pSUC2 without a signal peptide, and the YTK12 yeast strain without the vector as negative controls. After that, the grown yeast plaques were diluted with water to different gradient concentrations, inoculated on CMD/-W and YPRAA, and cultured at 30°C for 3 days. In addition, according to the principle that colorless TTC turns red after reduction, we cultured the yeast plaques grown after transforming YTK12 in CMD/-W liquid medium overnight, collected and resuspended them in the corresponding suspension [750 µL sterile water, 250 µL 10 mM acetic acid–sodium acetate buffer pH = 4.7, 500 µL 10% sucrose solution (w/v)]. After that, the supernatant was added to 0.1% TTC solution, and the color change was recorded by photography.

Plant cell death (PCD) detection

To detect whether the expression of Aa049 can cause PCD, we used the ready-to-use seamless cloning kit (Sangon Biotech, Shanghai, China) to insert the full-length CDS of Aa049, the Aa049 without the signal peptide, and the gene fragment of Aa049 replaced with the tobacco signal peptide PR1 into the pICH31160 vector, respectively. Then, Agrobacterium tumefaciens GV3101 containing the plasmid pJIC SA_Rep was transformed. We used INF1 as a positive control for inducing cell death and an empty vector as a negative control. The OD₆₀₀ of all Agrobacterium solutions was adjusted to 0.8 and injected into the same tobacco leaf. The leaf status of the injection area was observed within one week. A total of 6 tobacco leaves were injected.

Creation of mutant strains and detection of effects on growth and virulence

To analyze the effects of Aa049 on the growth and pathogenicity of A. alternata, we used genetic recombination to replace the full-length gene fragment of Aa049 with the hygromycin gene HPH. First, we used the DNA of F20 as a template and PCR amplified the 5' and 3' flanking segments of Aa049 using primer pairs ΔAa049-F1/R1 and ΔAa049-F2/R2, respectively. At the same time, using vector pMDC43 (containing the HPH resistance gene fragment) as a template, PCR amplification was performed using primer pair ΔAa049-F3/R3 to obtain the HPH gene fragment, and then PCR amplification using primers ΔAa049-F3/R2 to obtain HPH + 3' flanking segments. Finally, PCR amplification using primers ΔAa049-F4/R2 obtained the gene fragment for replacement, which included the 5' and 3' flanking segments and HPH. Afterwards, the recombinant gene fragment was transformed into the protoplasts of A. alternata by the PEG-mediated method (Liu, et al. 2020) and then applied to a PDA medium containing hygromycin to screen ΔAa049 mutant strains. The single colonies grown were confirmed by PCR and qRT-PCR methods. To observe the effect of Aa049 mutation on the morphology and growth, we inoculated the mutant and wild-type strains with the same size of fungal cake on PDA at the same time and placed them at 28°C for 7 days (5 PDA plates for each strain). Afterwards, the diameter of the colonies of each strain was counted, and the morphology of the colonies was photographed. To observe the effect of Aa049 mutation on hyphae growth, we took hyphae grown for 7 days of wild-type strains and mutants, and placed them under a DM1000 light microscope (Leica, Wetzlar, Germany) for observation. To assess the effect of Aa049 mutation on the pathogenicity of A. alternata, we inoculated wild-type and ΔAa049 mutant strains on chrysanthemums according to the inoculation method described above. 2 days later, the affected area was counted.

Congo red staining

To detect the effect of the loss of Aa049 on the integrity of the cell wall of A. alternata, we used Congo red dye to stain the mycelia of the wild-type and mutant strains. Precisely, 0.1 g of Congo red dye was weighed and dissolved in 10 mL of ddH₂O to prepare a solution. Mycelia grown for 7 days were placed in the dye solution for 15 minutes. After that, the mycelia were rinsed with water and a small amount of mycelia was placed on a LSM800 confocal laser scanning microscope (Zeiss, Oberkochen, Germany) to observe the red fluorescence.

Inoculation treatment after transient overexpression of Aa049 in tobacco

To explore whether Aa049 promotes the infection of A. alternata, we injected Agrobacterium solution containing an empty vector or p35S::Aa049 (OD₆₀₀ = 0.8) on the left and right sides of the same N. benthamiana leaf, cultured it in a dark environment at 22°C for one day, and then inoculated A. alternata on the back of the leaves injected with Agrobacterium solution according to the inoculation method described above. After 2 days, the leaves were photographed, and the lesion area was counted. A total of 7 N. benthamiana leaves were treated.

Creation of Aa049 overexpression transgenic chrysanthemum

To obtain Aa049 overexpression lines in chrysanthemum, we used the Aa049 cloning vector as a template and inserted the PCR amplification product into the pORE-R4-35AA vector through the Sma I and Cla I cloning sites. The above successfully constructed vectors was transformed into A. tumefaciens EHA105, and the transgenic plants were further obtained by Agrobacterium-mediated leaf disc transformation (Li et al., 2015). Then, RNA was extracted according to the method described above, then reverse transcribed into cDNA, and the positive lines were detected at the transcription level by semi-quantitative PCR.

NBT staining

The inoculated and uninoculated chrysanthemum leaves were put in NBT solution (1mg/mL; pH = 7.8) and stained under light for 12 hours. Then, they were transferred into 80% and 95% alcohol, respectively, and decolorized in boiling water for 15 minutes. Then, the leaves were put in 100% alcohol and stored at 4°C for photography.

Determination of ROS-related biochemical indicators

Chrysanthemum leaves inoculated with wild-type F20 and mutant strains were sampled, quickly frozen in liquid nitrogen, and then stored at -80°C. CAT and SOD activities, the ability to produce superoxide anions were determined using Commercial Assay Kits purchased from Nanjing Jiancheng Company. The operation was carried out strictly following the instructions, and each experiment was repeated three times.

Data processing and analysis

The lesion area was measured and calculated using ImageJ software (NIH, Bethesda, MD, USA). All data were analyzed using IBM SPSS Statistics 20 (IBM Corp., Armonk, NY, USA). The significant difference between any two groups of data was analyzed using Student’s t-test (*, P < 0.05; **, P < 0.01), and the significant difference between 3 or more groups of data was analyzed using Duncan’s multiple test (P < 0.05).

Bioinformatics analysis of Aa049

In the preliminary phase, we identified a predicted GPI-AP in a transcriptome database, in which chrysanthemum leaves were infected with A. alternata (Liu, et al. 2020). We cloned its ORF from A. alternata, which spans 489 bp and encodes 162 amino acids. Due to its amino acid sequence matching that of the gene locus_tag B0T12DRAFT_157049 in A. alternata according to NCBI-BLAST, we designated it Aa049. SignalP 6.0 predicted the presence of a signal peptide at positions 1–20 of the N-terminal of its amino acid sequence (Fig. 1A, B). Prediction by BIG-PI Fungal Predictor indicated amino acid residues at positions 141 and 142 as potential GPI modification sites (Fig. 1B). TMHMM 2.0 predicted that Aa049 lacks transmembrane structure (Fig. 1C). Evolutionary analysis revealed Aa049 to be most closely related to a homologous protein in Alternaria postmessia (Fig. 1D). MEME software identified 6 conserved motifs within the amino acid sequence of Aa049 (Fig. S1), suggesting conservation particularly among fungi of the Alternaria genus, with motifs 2, 3, 4, and 6 being conserved across different fungal species (Fig. S1). These results implied that Aa049 may play a crucial role in the interaction between fungi and plants.

Expression pattern of Aa049 during A. alternata invasion

To analyze the expression pattern of Aa049 during A. alternata invasion of chrysanthemum, we conducted qRT-PCR by sampling chrysanthemum leaves inoculated with A. alternata at different time points. The results showed that compared to 0 hours post-inoculation (hpi), Aa049 showed a progressively downregulated expression trend during pathogen infection (Fig. 2), which suggestd that Aa049 may be suppressed by immune-related genes in the plant.

The signal peptide of Aa049 exhibits secretion

According to previous predictions by the SignalP6.0 software, a signal peptide is present at the N-terminus (positions 1–20) of its amino acids. To investigate whether the signal peptide of Aa049 exhibits secretion, we constructed vectors pSUC2::Aa049-FL (full-length amino acids) and pSUC2::Aa049-SP (only containing the signal peptide) and transformed them into the yeast strain YTK12. This strain lacks the gene for invertase and cannot grow in a tryptophan-deficient environment. The pSUC2 vector can synthesize tryptophan but lacks the signal peptide. Only when a secretion-functional signal peptide is present can promote invertase to be secreted to convert raffinose into glucose, which yeast can utilize, enabling the YTK12 strain to grow on the YPRAA medium. In this experiment, we used pSUC2::Avr1b as a positive control, and pSUC2::Mg87 (non-secretion-active), empty pSUC2 vector without a signal peptide, and untransformed YTK12 yeast strain as negative controls. Results showed that on the CMD/-W medium, all transformed strains grew normally except the untransformed YTK12 strain, indicating that all vectors were successfully transformed into YTK12. Meanwhile, on the YPRAA medium, only Aa049-FL and Aa049-SP grew normally like the positive control. All negative controls showed inhibited growth (Fig. 3). The yeast secretion assay indicated that the signal peptide of Aa049 exhibits secretion. Furthermore, the TTC assay showed that the solution containing pSUC2::Aa049-FL or pSUC2::Aa049-SP, like the positive control, turned red, while the rest remained colorless (Fig. 3). This further confirmed that the signal peptide of Aa049 exhibits secretion, allowing invertase to be secreted into the solution to convert raffinose or sucrose into reducible glucose, thereby reducing TTC to the red triphenylformazan (TTF). Thus, both the yeast secretion assay and the TTC assay demonstrated that the signal peptide of Aa049, consisting of amino acids 1–20 at the N-terminus, exhibits secretion.

Transient overexpression of Aa049 induces localized plant cell death, dependent on native signal peptide

Previous studies have shown that the expression of GPI-APs can cause cell death in plants (Hu, et al. 2023). To determine whether Aa049 can induce PCD, we constructed vectors pICH31160-Aa049-FL-3HA, pICH31160-Aa049-ΔSP-3HA, and pICH31160-SP^PR1-Aa049-3HA. These vectors were transformed into A. tumefaciens strain GV3101 and then transiently transformed into N. benthamiana. It was observed that the full-length Aa049 induced cell death in tobacco, while Aa049 without the signal peptide and Aa049 with the signal peptide replaced by PR1 only caused chlorosis in tobacco leaves but did not induce cell death (Fig. 4). These results indicated that the ability of Aa049 to induce PCD depends on its native signal peptide.

Transient overexpression of Aa049 in tobacco suppresses the expression of immune genes

Since the expression of Aa049 in tobacco can cause cell death, we investigated whether it also induces the expression of plant immunity-related genes. We performed qRT-PCR analysis on tobacco leaves sampled 24 hours after infiltration with Agrobacterium containing either the empty vector or p35S::Aa049. The results showed that the expression levels of several defense-related genes, including NbHSR203J, NbGLNb, NbPR1, NbPR2, NbBAK1, and NbWRKY8, were downregulated in the leaves of transient overexpression of Aa049 (Fig. 5). These suggestd that Aa049 may act as a virulence factor, inhibiting the plant's immune response.

Overexpression of Aa049 in plants promotes A. alternata infection

To investigate whether the expression of Aa049 promotes pathogen invasion, we first infiltrated tobacco with Agrobacterium suspension containing either p35S::Aa049 or an empty vector, followed by inoculation with A. alternata. Compared to the control, transient overexpression of Aa049 resulted in more severe disease symptoms and significantly larger lesion areas on the tobacco leaves (Fig. 6A, B). Furthermore, we constructed transgenic chrysanthemum lines overexpressing Aa049 (OE-Aa049) (Fig. S2) and conducted infection assays. Compared to wild-type plants, OE-Aa049 transgenic chrysanthemum leaves were more susceptible to A. alternata. Two days post-inoculation, lesions expanded along the leaf veins beyond the inoculation sites (Fig. 6C), with a marked increase in lesion size (Fig. 6D). These results indicated that overexpression of Aa049 in both tobacco and chrysanthemum enhanced susceptibility to A. alternata and Aa049 functioned as a virulence factor.

A, Representative image of the disease situation of tobacco plants inoculated with A. alternata 2 days after transient overexpression of Aa049. Bar = 1 cm. B, Lesion area in tobacco, n = 7. C, Representative image of the disease situation of wild-type ‘Jinba’ and transgenic chrysanthemums overexpressing Aa049 inoculated with A. alternata 2 days after inoculation. Bar = 1 cm. D, Lesion area in chrysanthemums, n = 9. All statistical analyses were performed using Student’s t-test (**, P < 0.01).

Mutation of Aa049 does not affect the growth and morphology, but reduces the virulence of A. alternata

To determine whether Aa049 contributes to the growth of A. alternata, we constructed the deletion mutant strain (ΔAa049) using PEG-mediated gene recombination. In this method, the full-length coding sequence of Aa049 in the wild-type strain F20 (F20-WT) was replaced with the hygromycin resistance gene HPH (Fig. 7A). We identified three positive transformants through PCR and qRT-PCR (Fig. 7B, C). Compared to F20-WT, the ΔAa049 mutant strains showed no changes in the colonial and hyphal morphology of A. alternata (Fig. S3A, B). Measurement of colony diameters after one week of growth revealed that the colony diameter of the ΔAa049 mutants was similar to that of F20-WT (Fig. S3C), indicating that the mutation of Aa049 does not affect the growth rate of hyphae. These results demonstrated that the deletion of Aa049 does not affect the growth and morphology of A. alternata. GPI-APs in fungi have been reported to participate in the structural composition of fungal cell walls (Nather and Munro 2008). To explore whether Aa049 is also involved in this biological process, we stained and observed F20-WT and the ΔAa049 mutant strains with Congo red. Fungal cell walls contain major components such as β-1,3-glucan, cellulose, and chitin. Congo red has a high affinity for β-1,3-glucan and fluoresces to indicate the distribution of cell wall components. After staining with Congo red, the hyphae of F20-WT displayed uniform fluorescence labeling, while the hyphae of the ΔAa049 mutant strains showed uneven fluorescence distribution with sporadic spots (Fig. S3D). These results indicated that mutation of Aa049 disrupts the normal structure of the cell wall of A. alternata.

To assess whether Aa049 contributes to the virulence of A. alternata, we inoculated the chrysanthemum cultivar 'Jinba' with F20-WT or ΔAa049 mutant strains. After 2 days post-inoculation, we observed that hyphae of F20-WT had extensively penetrated the chrysanthemum leaves, with abundant growth on the leaf surfaces. In contrast, infection symptoms were significantly reduced in chrysanthemums inoculated with the ΔAa049 mutant strains, with only sparse hyphae present on the leaf surfaces (Fig. 7D). Additionally, the lesion areas were noticeably smaller (Fig. 7E). These inoculation results indicated that Aa049 plays a significant role in the pathogenicity of A. alternata.

Aa049 may suppress plant immunity via the ROS pathway

ROS burst is a hallmark of plant PTI (pattern-triggered immunity) response. To investigate whether Aa049 induces changes in the expression of genes involved in the ROS pathway during A. alternata invasion, we conducted qRT-PCR analysis on the expression of genes encoding ROS synthesis and ROS scavenging enzymes. The results showed that compared to chrysanthemum leaves inoculated with F20-WT, those inoculated with the ΔAa049 mutant strains exhibited downregulation of ROS synthesis-related genes CmRBOHC and CmRBOHD, while ROS scavenging-related genes such as CmAPX6, CmCAT, and CmSOD were upregulated (Fig. 8A). Additionally, staining with Nitrotetrazolium Blue chloride (NBT) on inoculated leaves revealed lighter staining in leaves inoculated with the ΔAa049 mutant strains compared to those inoculated with F20-WT, indicating lower production of superoxide anions during A. alternata infection due to the absence of Aa049 (Fig. 8B). Furthermore, quantitative measurements of ROS-related markers in inoculated chrysanthemum leaves showed lower levels of superoxide anions and higher levels of CAT and SOD activities in leaves inoculated with the ΔAa049 mutant strains (Fig. 8C-E). These results suggest that Aa049 may manipulate plant immune responses by modulating ROS synthesis and scavenging pathways.

Aa049, as a widely distributed GPI-anchored protein in Alternaria Nees, participates in pathogenesis

In the prolonged struggle between pathogens and plants, numerous effective pathogenic strategies have evolved (Wang, et al. 2022). GPI-APs, as crucial components of pathogen cell walls, play a key role in protecting pathogens from environmental stresses (Liu, et al. 2020). This study identified a GPI-AP in A. alternata for the first time, with bioinformatics analysis revealing a signal peptide region at its N-terminus and a GPI anchoring site at its C-terminus (Fig. 1). The results of Congo Red staining indicated defective cell wall structures in its mutants (Fig. 8), suggesting that Aa049 likely functions as a cell wall GPI-AP and contributes significantly to the integrity of the cell wall of A. alternata. Infection assays demonstrated significantly reduced virulence in mutant strains lacking Aa049 (Fig. 9), further illustrating the importance of Aa049 as a constituent of the cell wall of A. alternata that affects its infectivity. Amino acid sequence analysis of Aa049 shows conservation across Alternaria fungi (Fig. S1), indicating its role as a conserved virulence factor widely present in Alternaria species. The second, third, fourth, and sixth motifs are conserved across different fungal species (Fig. S1), suggesting potential vital roles in other pathogenic fungi, though specific mechanisms require further research.

Aa049 relies on its native signal peptide to induce plant cell death

Cell death, as a kind of plant hypersensitivity responses (HR), is a measure taken by plants to achieve self-protection when they are subjected to external biological stress, which can effectively limit the further reproduction of pathogens in plants, so biotrophs and hemi-biotrophs have evolved a series of mechanisms to inhibit HR to evade host detection and inhibit the activation of plant immunity. For example, several specific effectors secreted by Pseudomonas syringae pv. tomato DC3000, Xanthomonas campestris pv. Vesicatoria, Colletotrichum fructicola, and oomycetes can enter plant cells through different secretion systems and inhibit the occurrence of HR, thereby weakening plant immunity (Bos, et al. 2006; Fujikawa, et al. 2006; Guo, et al. 2009; Jamir, et al. 2004; Kelley, et al. 2010; Shang, et al. 2024). As a pathogenic fungus that feeds on dead tissues, the strategy of attack evolved by A. alternata in the long-term struggle with plants may mainly aim to promote plant tissue death. Previous studies on the necrotrophic fungi Botrytis cinerea and S. sclerotiorum have shown that these pathogens both release some cell death-inducing proteins (CDIPs), such as PG1/PG2 (Leisen, et al. 2022), SsNep1, SsNep2 (Dallal Bashi, et al. 2010), and SsNE2 (Seifbarghi, et al. 2020) to infect a large number of different host plants to promote better colonization. Most of these proteins are secreted proteins, and a GPI-AP, SsGSR1, has recently been reported in S. sclerotiorum as a CDIP (Hu, et al. 2023). This study revealed for the first time that Aa049 from Alternaria spp., as a GPI-AP, can cause local cell death when it is overexpressed in tobacco, and further inoculation experiments showed that plant tissues overexpressing Aa049 were more sensitive to A. alternata, indicating that this necrosis reaction did promote the infection of A. alternata. This may be due to the abundant nutrients provided by the necrotic tissues to A. alternata, which is consistent with the conclusions of previous studies on the pathogenesis of necrotrophs. Specifically, only the full-length Aa049 (including signal peptide) protein could induce PCD, while the removal of the signal peptide and the replacement with tobacco signal peptide only caused yellowing and shrinkage of plants, indicating that the signal peptide of Aa049 not only has the role of guiding secretion, but may also have other special functions. It may have an important contribution to promoting virulence in the process of A. alternata infection, and the related mechanism needs to be further studied.

Pathogens are pathogenic through the ROS pathway

Reactive oxygen species play a crucial role in defending against external stresses as the main immune defense substances and important signaling components plants release during biotic and abiotic stresses (Ding, et al. 2022). When infected by pathogens, plants produce more ROS at the site of infection to kill pathogens. In addition, ROS burst can easily cause damage and even necrosis of plant host cells, which is considered one of the important markers of HR. As a result, biotrophs and hemi-biotrophs have evolved strategies to prevent the high production and accumulation of ROS in plants or to enhance the removal of ROS. However, unlike biotrophs and hemi-biotrophs, necrotrophs allow the host plant to produce and accumulate more ROS, because this process promotes the occurrence of HR for better invasion and colonization of pathogens, such as B. cinerea, S. sclerotiorum and Leptosphaeria maculans (Govrin and Levine 2000; Heller and Tudzynski 2011; Li, et al. 2008). In addition, it has been found that Colletotrichum coccodes activates ROS burst by secreting ammonium ions, thereby triggering PCD and promoting the virulence to tomato (Alkan, et al. 2009). In this study, it was found that the ROS synthesis-related genes were significantly down-regulated in chrysanthemum leaves inoculated with ΔAa049 mutants compared with wild-type strains, and on the contrary, the ROS clearance genes were significantly up-regulated. Further, NBT staining and the determination of related physiological indicators were also consistent with the results of qRT-PCR. These results indicate that Aa049 is likely to accelerate the infection of pathogens by breaking the ROS homeostasis in plants, promoting the PCD process, and disrupting the host immune system. This is the first time to reveal a relationship between GPI-APs in A. alternaria and plant ROS pathway, and this result also provides important clues for subsequent research on plant-microbial interactions.

In this study, we have for the first time identified a GPI-AP, Aa049, in A. alternata, which is able to trigger PCD depending on its native signal peptide. The overexpression of Aa049 promoted the infection of A. alternata, and the mutant strains showed reduced virulence, indicating that Aa049 has an important contribution to the virulence of A. alternata. Congo red staining results suggested that Aa049 plays a role as an essential component of the cell wall of pathogen. Moreover, the integrity of the cell wall is closely related to the invasion and colonization of A. alternata. More profoundly, we speculated that the pathogenic mechanism of Aa049 may be related to the synthesis and scavenging of ROS in plants. All these results indicated that Aa049 plays a role as an important pathogenic factor evolved in the long-term struggle between pathogens and plants, and can be considered as an important target in future disease resistance breeding and biopesticide production.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This research was funded by the National Natural Science Foundation of China (32171854), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Author contribution

statement B.D., J.J., S.C., and W.F. conceived the study and designed the experiment. B.D., Y.S., and J.Z. performed the experiment. B.D. analyzed the data. B.D. and J.J. wrote the article. F.C., W.F., and Z.G. ensured financial support. B.D. and J.J. revised the manuscript.

Acknowledgments

We would like to thank Prof. Haitao Cui (Fujian Agriculture and Forestry University), and Prof. Suomeng Dong (Nanjing Agricultural University) for their kind contributions to the vectors used in this experiment.

Alkan N, Davydov O, Sagi M, Fluhr R, Prusky D (2009) Ammonium secretion by Colletotrichum coccodes activates host NADPH oxidase activity enhancing host cell death and fungal virulence in tomato fruits. Mol plant-microbe interactions: MPMI 22:1484–1491
Bos JI, Kanneganti TD, Young C, Cakir C, Huitema E, Win J, Armstrong MR, Birch PR, Kamoun S (2006) The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana. Plant journal: cell Mol biology 48:165–176
Cho Y (2015) How the necrotrophic fungus Alternaria brassicicola kills plant cells remains an enigma. Eukaryot Cell 14:335–344
Cho Y, Kim KH, La Rota M, Scott D, Santopietro G, Callihan M, Mitchell TK, Lawrence CB (2009) Identification of novel virulence factors associated with signal transduction pathways in Alternaria brassicicola. Mol Microbiol 72:1316–1333
Cho Y, Ohm RA, Grigoriev IV, Srivastava A (2013) Fungal-specific transcription factor AbPf2 activates pathogenicity in Alternaria brassicicola. Plant journal: cell Mol biology 75:498–514
Cho Y, Srivastava A, Ohm RA, Lawrence CB, Wang KH, Grigoriev IV, Marahatta SP (2012) Transcription factor Amr1 induces melanin biosynthesis and suppresses virulence in Alternaria brassicicola. PLoS Pathog 8:e1002974
Chun J, Ko YH, So KK, Cho SH, Kim DH (2022) A fungal GPI-anchored protein gene functions as a virulence and antiviral factor. Cell Rep 41:111481
Crudo F, Varga E, Aichinger G, Galaverna G, Marko D, Dall'Asta C, Dellafiora L (2019) Co-Occurrence and Combinatory Effects of Alternaria Mycotoxins and other Xenobiotics of Food Origin: Current Scenario and Future Perspectives. Toxins 11
Dallal Bashi Z, Hegedus DD, Buchwaldt L, Rimmer SR, Borhan MH (2010) Expression and regulation of Sclerotinia sclerotiorum necrosis and ethylene-inducing peptides (NEPs). Mol Plant Pathol 11:43–53
DeMers M (2022) Alternaria alternata as endophyte and pathogen. Microbiology 168
Ding LN, Li YT, Wu YZ, Li T, Geng R, Cao J, Zhang W, Tan XL (2022) Plant Disease Resistance-Related Signaling Pathways: Recent Progress and Future Prospects. Int J Mol Sci 23
Duhan D, Gajbhiye S, Jaswal R, Singh RP, Sharma TR, Rajarammohan S (2021) Functional Characterization of the Nep1-Like Protein Effectors of the Necrotrophic Pathogen - Alternaria brassicae. Front Microbiol 12:738617
Enjoji SJJoPP (1931) Two new diseases of cineraria I. Ring spot disease 18:428–432
Fernandes C, Casadevall A, Gonçalves T (2023) Mechanisms of Alternaria pathogenesis in animals and plants. FEMS Microbiol Rev 47
Fujikawa T, Ishihara H, Leach JE, Tsuyumu S (2006) Suppression of defense response in plants by the avrBs3/pthA gene family of Xanthomonas spp. Mol plant-microbe interactions: MPMI 19:342–349
Fujita M, Kinoshita T (2012) GPI-anchor remodeling: potential functions of GPI-anchors in intracellular trafficking and membrane dynamics. Biochim Biophys Acta 1821:1050–1058
Govrin EM, Levine A (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea. Curr biology: CB 10:751–757
Guo M, Tian F, Wamboldt Y, Alfano JR (2009) The majority of the type III effector inventory of Pseudomonas syringae pv. tomato DC3000 can suppress plant immunity. Mol plant-microbe interactions: MPMI 22:1069–1080
Heller J, Tudzynski P (2011) Reactive oxygen species in phytopathogenic fungi: signaling, development, and disease. Annu Rev Phytopathol 49:369–390
Howard RJ, Ferrari MA, Roach DH, Money NP (1991) Penetration of hard substrates by a fungus employing enormous turgor pressures. Proc Natl Acad Sci USA 88:11281–11284
Hu Y, Gong H, Lu Z, Zhang P, Zheng S, Wang J, Tian B, Fang A, Yang Y, Bi C, Cheng J, Yu Y (2023) Variable Tandem Glycine-Rich Repeats Contribute to Cell Death-Inducing Activity of a Glycosylphosphatidylinositol-Anchored Cell Wall Protein That Is Associated with the Pathogenicity of Sclerotinia sclerotiorum. Microbiol Spectr 11:e0098623
Jamir Y, Guo M, Oh HS, Petnicki-Ocwieja T, Chen S, Tang X, Dickman MB, Collmer A, Alfano JR (2004) Identification of Pseudomonas syringae type III effectors that can suppress programmed cell death in plants and yeast. Plant journal: cell Mol biology 37:554–565
Kelley BS, Lee SJ, Damasceno CM, Chakravarthy S, Kim BD, Martin GB, Rose JK (2010) A secreted effector protein (SNE1) from Phytophthora infestans is a broadly acting suppressor of programmed cell death. Plant journal: cell Mol biology 62:357–366
Leisen T, Werner J, Pattar P, Safari N, Ymeri E, Sommer F, Schroda M, Suárez I, Collado IG, Scheuring D, Hahn M (2022) Multiple knockout mutants reveal a high redundancy of phytotoxic compounds contributing to necrotrophic pathogenesis of Botrytis cinerea. PLoS Pathog 18:e1010367
Li C, Barker SJ, Gilchrist DG, Lincoln JE, Cowling WA (2008) Leptosphaeria maculans elicits apoptosis coincident with leaf lesion formation and hyphal advance in Brassica napus. Molecular plant-microbe interactions: MPMI 21:1143–1153
Li L, Zhu XM, Zhang YR, Cai YY, Wang JY, Liu MY, Wang JY, Bao JD, Lin FC (2022) Research on the Molecular Interaction Mechanism between Plants and Pathogenic Fungi. Int J Mol Sci 23
Li Q, Feng Y, Li J, Hai Y, Si L, Tan C, Peng J, Hu Z, Li Z, Li C, Hao D, Tang W (2024) Multi-omics approaches to understand pathogenicity during potato early blight disease caused by Alternaria solani. Front Microbiol 15:1357579
Liu C, Xing J, Cai X, Hendy A, He W, Yang J, Huang J, Peng YL, Ryder L, Chen XL (2020) GPI7-mediated glycosylphosphatidylinositol anchoring regulates appressorial penetration and immune evasion during infection of Magnaporthe oryzae. Environ Microbiol 22:2581–2595
Liu Y, Xin J, Liu L, Song A, Guan Z, Fang W, Chen F (2020) A temporal gene expression map of Chrysanthemum leaves infected with Alternaria alternata reveals different stages of defense mechanisms. Hortic Res 7:23
Liu Y, Xin J, Liu L, Song A, Liao Y, Guan Z, Fang W, Chen F (2020) Ubiquitin E3 Ligase AaBre1 Responsible for H2B Monoubiquitination Is Involved in Hyphal Growth, Conidiation and Pathogenicity in Alternaria alternata. Genes 11
Lu K, Zhang M, Yang R, Zhang M, Guo Q, Baek KH, Xu H (2019) The MAP Kinase Kinase Gene AbSte7 Regulates Multiple Aspects of Alternaria brassicicola Pathogenesis. plant Pathol J 35:91–99
Nather K, Munro CA (2008) Generating cell surface diversity in Candida albicans and other fungal pathogens. FEMS Microbiol Lett 285:137–145
Pelser PB, Nordenstam B, Kadereit JW, Watson LEJT (2007) An ITS phylogeny of tribe Senecioneae (Asteraceae) and a new delimitation of. Senecio L 56:1077–1104
Qiu C, Halterman D, Zhang H, Liu Z (2024) Multifunctionality of AsCFEM6 and AsCFEM12 effectors from the potato early blight pathogen Alternaria solani. Int J Biol Macromol 257:128575
Rittenour WR, Harris SD (2013) Glycosylphosphatidylinositol-anchored proteins in Fusarium graminearum: inventory, variability, and virulence. PLoS ONE 8:e81603
Seifbarghi S, Borhan MH, Wei Y, Ma L, Coutu C, Bekkaoui D, Hegedus DD (2020) Receptor-Like Kinases BAK1 and SOBIR1 Are Required for Necrotizing Activity of a Novel Group of Sclerotinia sclerotiorum Necrosis-Inducing Effectors. Front Plant Sci 11:1021
Shang S, Liu G, Zhang S, Liang X, Zhang R, Sun G (2024) A fungal CFEM-containing effector targets NPR1 regulator NIMIN2 to suppress plant immunity. Plant Biotechnol J 22:82–97
Song T, Zhang Y, Zhang Q, Zhang X, Shen D, Yu J, Yu M, Pan X, Cao H, Yong M, Qi Z, Du Y, Zhang R, Yin X, Qiao J, Liu Y, Liu W, Sun W, Zhang Z, Wang Y, Dou D, Ma Z, Liu Y (2021) The N-terminus of an Ustilaginoidea virens Ser-Thr-rich glycosylphosphatidylinositol-anchored protein elicits plant immunity as a MAMP. Nat Commun 12:2451
Srivastava A, Ohm RA, Oxiles L, Brooks F, Lawrence CB, Grigoriev IV, Cho Y (2012) A zinc-finger-family transcription factor, AbVf19, is required for the induction of a gene subset important for virulence in Alternaria brassicicola. Molecular plant-microbe interactions: MPMI 25:443–452
Takano KJJJoP (2001) Leaf spot of dusty-miller caused by Alternaria cinerariae Hori et Enjoji [Abstract]. 167
Wang C, Wang J, Zhang D, Cheng J, Zhu J, Yang Z (2023) Identification and functional analysis of protein secreted by Alternaria solani. PLoS ONE 18:e0281530
Wang C, Zhang D, Cheng J, Zhao D, Pan Y, Li Q, Zhu J, Yang Z, Wang J (2022) Identification of effector CEP112 that promotes the infection of necrotrophic Alternaria solani. BMC Plant Biol 22:466
Wang Y, Pruitt RN, Nürnberger T, Wang Y (2022) Evasion of plant immunity by microbial pathogens. Nat Rev Microbiol 20:449–464
Yago JI, Lin CH, Chung KR (2011) The SLT2 mitogen-activated protein kinase-mediated signalling pathway governs conidiation, morphogenesis, fungal virulence and production of toxin and melanin in the tangerine pathotype of Alternaria alternata. Mol Plant Pathol 12:653–665

Download PDF

Reviewers agreed at journal
21 Aug, 2024
Reviewers invited by journal
06 Aug, 2024
Editor assigned by journal
06 Aug, 2024
First submitted to journal
03 Aug, 2024

You are reading this latest preprint version

A Glycosylphosphatidylinositol-anchored protein from Alternaria alternata triggers cell death and negatively modulates immunity responses in chrysanthemum

Status:

Version 1

Abstract

Figures

Key Message

Introduction

Materials and methods