Fig 1.
Identification of Alternaria alternata effector AaAlta1.
(A) Schematic diagram of AaAlta1. The 159 amino acid protein has an N-terminal signal peptide (SP) of 16 amino acids for secretion of the mature effector, the A. alternata allergen-1 motif sequence. (B) AaAlta1 induced cell death in Nicotian benthamiana and chrysanthemum. Representative N. benthamiana leaves directly infiltrated with purified AaAlta1 protein (300 nM to 3 µM) and photographed 2 days post-infiltration. β-glucuronidase (GUS; control, purified in the same way as AaAlta1) is the culture supernatant control. The image on the right shows chrysanthemum leaves directly infiltrated with purified AaAlta1 and photographed 3 days post-infiltration. (C) An SP is required for AaAlta1-induced cell death. Nicotiana benthamiana leaves infiltrated with the Agrobacterium tumefaciens strain GV3101 cells harboring the genes coding for A. alternata effector proteins, Phytophthora infestans INF1 gene, and empty vector (EV). The Agrobacterium cells harboring the INF1 gene and EV were used as positive and negative controls, respectively. Images were captured at 7 days. (D) Western blot analysis of the transiently expressed proteins obtained from the three agroinfiltrated leaves was performed using an anti-hemagglutinin (HA) antibody. Actin was an equal loading control. (E) Luminol-based assay of reactive oxygen species (ROS) burst in the leaves of N. benthamiana treated with 1 μM Alta1. Leaves treated with 200 nM flg22 were used as a positive control, whereas those treated with H2O and 1 µM GUS were used as a negative control. (F) Yeast secretion trap assay to verify the functionality of SP in AaAlta1. The yeast suc2 mutant strain cannot utilize sucrose as a carbon source because of the lack of functional invertase but can use glucose. Colonies were spotted on synthetically defined YPRAA (a medium containing 1% yeast extract, 2% peptone, 2% raffinose, 2 μg/mL antimycin A, and 2% agar) plates with sucrose or glucose and antimycin A. The vector control (EV) and YTK12 were used as a negative control. Pathogenesis-related (PR) 1 represents the SP from the PR1 protein and was used as a positive control. The yeast suc2 mutant strain expressing pSUC2-Alta1SP was resuspended in 0.1% TTC (2,3,5-triphenyltetrazolium chloride) solution and checked for the formation of red color. (G) Relative expression levels of AaAlta1 during infection were assessed by quantitative reverse transcriptase polymerase chain reaction. Data are presented as the mean ± standard error of three biological replicates. Different letters at the top of error bars indicate significant differences (P < 0.05, Tukey’s test).
Fig 2.
AaAlta1 is important for the expression of the full virulence of Alternaria alternata.
(A) Radial growth of A. alternata (WT, control), Δalta1 (AaAlta1 knockout mutant), and Δalta1/Alta1 (complementation) strains on potato dextrose agar plates. All indicated fungal strains were grown for 2 and 7 days at 28 °C. The lower panel shows the conidiophore morphology of strains grown for 2 days. The upper panel shows the conidiophore morphology of strains grown for 7 days. (B) Symptoms of the black spot disease on chrysanthemum leaves inoculated with WT, Δalta1 mutant, and complemented strain Δalta1/Alta1. Images were captured at 48 h post-inoculation (hpi). Scale bar= 1 cm. (C) Disease severity was determined by measuring the lesion area (mm2) of leaves 48 hpi. Data are presented as the mean ± standard error of four biological replicates. Different letters at the top of error bars indicate significant differences (P < 0.05, Tukey’s test).
Fig 3.
AaAlta1 induces chrysanthemum resistance to Alternaria alternata.
(A) The disease symptoms of A. alternata in chrysanthemum leaves infiltrated with buffer (Mock) or 300 nM Alta1. Images were captured at 48 hpi. Scale bar=1 cm. (B) Mean lesion size on Alta1-infiltrated and mock-infiltrated chrysanthemum inoculated with A. alternata at 48 hpi. Data are presented as the mean ± standard error of four biological replicates. * P ≤ 0.05 compared to control, as calculated by one-way analysis of variance (ANOVA). (C) Volcano plot showing upregulated (red) and downregulated (blue) genes in the Alta1-infiltrated (Alta1) leaf samples compared with that in the mock-infiltrated (Mock) leaf samples for 24 h. (D) Kyoto Encyclopedia of Genes and Genomes enrichment analysis of upregulated differentially expressed genes from Alta1- versus mock infiltrated tissues. The top 20 pathways with the most significant P values are shown. (E) Heat map of gene expression associated with disease resistance in the transcriptomes. The gene expression values are normalized log2(FPKM [fragments per kilobase of transcript per million fragments mapped] + 1). From left to right is Mock and Alta1. Mock, mock-infiltrated group; Alta1, 300 nM Alta1-infiltrated group. (F) Quantitative reverse transcriptase polymerase chain reaction analysis of MYC2, WRKY33, RbohD and PDF1.2 genes in mock and Alta1-infiltrated leaves under normal growth conditions. Data are presented as the mean ± standard error of three biological replicates. *P ≤ 0.05 compared with control, as calculated by one-way ANOVA. (G) Measurements of jasmonic acid (JA) content in chrysanthemum leaves infiltrated with 300 nM Alta1 or mock solution. Data are presented as the mean ± standard error of four biological replicates. *P ≤ 0.05 compared with control, as calculated by one-way ANOVA.
Fig 4.
AaAlta1 physically associates with CmWD40.
(A) Results of the yeast-two-hybrid assay show that AaAlta1 interacts with CmWD40. (B) Pull-down assays were performed to test whether AaAlta1 interacts with CmWD40 in vitro. The GST-CmWD40 fragment was used to pull down His-Alta1 with the empty GST fragment as a negative control. The GST-CmWD40/His-Alta1 and GST/His-Alta1 complexes were incubated and detected using an anti-His antibody after washes. TheHis-Alta1 could be pulled down using GST-CmWD40 (see the lane designated GST-CmWD40 output) but not using the GST control fragment (see the lane designated GST output). (C) A bimolecular fluorescence complementation assay confirmed the interaction between AaAlta1 and CmWD40. YFP: yellow fluorescent protein; mCherry: nuclear localization shown using red fluorescent protein (RFP) activity; DIC: differential interference contrast image; merge: overlay of YFP, RFP, and DIC images.
Fig 5.
CmWD40 regulates the resistance of chrysanthemum plants to Alternaria alternata.
(A) The rhythmic expression pattern of CmWD40 and the temporal variation in chrysanthemum susceptibility to A. alternata. The line chart represents the rhythmic expression pattern of CmWD40 in wild-type (WT) plants under long-day (LD) conditions. Leaves were collected every 6 h at the indicated times, and gene expression levels were assessed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The bar chart represents the mean lesion size on chrysanthemum leaves inoculated with A. alternata at dawn, i.e., at zeitgeber time (ZT)0 and every 6 h for 48 h under LD conditions. The size of the lesions was statistically analyzed at 48 hpi. Data are presented as the mean ± standard error of three biological replicates. (B) Relative expression levels of the CmWD40 gene during infection were assessed using qRT-PCR. Data are presented as the mean ± standard error of three biological replicates. Different letters at the top of error bars indicate significant differences (P < 0.05, Tukey’s test). (C) Disease symptoms caused by A. alternata infection in chrysanthemum leaves. Leaves of WT and CmWD40 transgenic lines were inoculated with A. alternata, and images were captured at 48 hpi; scale bar=1 cm. (D) Identification of the CmWD40 overexpression (OX) transgenic line (OX-CmWD40) at the transcript level using qRT-PCR. Samples were collected at dawn. Different letters at the top of error bars indicate significant differences (P < 0.05, Tukey’s test). (E) Immunoblot analysis of CmWD40 in two transgenic lines overexpressing CmWD40. Samples were collected at dawn. Total proteins of transgenic seedlings were extracted and detected with anti-Flag antibodies. Actin was used as an equal loading control. (F) Mean lesion size on WT and OX-CmWD40 chrysanthemum leaves inoculated with A. alternata at 48 hpi. Data are presented as the mean ± standard error of three biological replicates. * P ≤ 0.05 compared with control, as calculated by one-way analysis of variance (ANOVA). (G) Expression of CmWD40 in CaLCuV-amiR-CmWD40 transgenic lines. Samples were collected at dawn. Different letters at the top of error bars indicate significant differences (P < 0.05, Tukey’s test).(H) Disease symptoms caused by A. alternata infection in chrysanthemum leaves. Leaves of the WT and cabbage leaf-curl geminivirus vector (CaLCuV)-amiR-CmWD40 lines were inoculated with A. alternata, and images were captured at 48 hpi; scale bar=1 cm. (I) Mean lesion size on WT and CmWD40 silenced chrysanthemum leaves inoculated with A. alternata at 48 hpi. Data are presented as the mean ± standard error of three biological replicates. * P ≤ 0.05 compared with control, as calculated by one-way ANOVA. (J) qRT-PCR analysis for expression of PDF1.2, WRKY33, and MYC2 genes reveals that their transcript levels are upregulated in OX-CmWD40 and CaLCuV-amiR-CmWD40 transgenic lines, respectively, upon infection with A. alternata. Data are presented as the mean ± standard error of three biological replicates. Different letters at the top of error bars indicate significant differences (P <0.05, Tukey’s test).
Fig 6.
CmWD40 activates JA biosynthesis and signaling to promote immunity.
(A) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes (DEGs) in the overexpression (OX)-CmWD40 transgenic line compared with that in the wild type (WT). The top 20 pathways with the most significant P-value are shown. (B) KEGG analysis of DEGs in the OX-CmWD40 transgenic line compared with that in the WT after Alternaria alternata infection for 48 h. The top 20 pathways with the most significant P-value are shown. (C) Gene Ontology (GO) analysis of DEGs in the OX-CmWD40 transgenic line compared with that in the WT after A. alternata infection for 48 h. The top 20 pathways with the most significant P-value are shown. (D) Heat map of the normalized log2(FPKM [fragments per kilobase of transcript per million fragments mapped] + 1) values of JA-related genes among the upregulated DEGs. WT, control, non-infected plants (WT ‘Jinba’); OX-WD40, non-infected OX-CmWD40 transgenic plants; WT-I, control, infected plants; OX-WD40-I, infected OX-CmWD40 transgenic plants. (E) Comparison of JA content in WT and OX-CmWD40 transgenic plants with or without A. alternata infection. Data are presented as the mean ± standard error of three biological replicates. * P ≤0.05 compared with control, as calculated by one-way ANOVA. (F) Expression levels of genes implicated in JA biosynthesis and signal transduction in different samples. The left vertical axis represents the relative gene expression levels from qRT-PCR (orange), and the right vertical axis represents the FPKM values obtained from RNA-seq (green). (G) Expression levels of defense-related genes in different samples. The left vertical axis represents the relative gene expression levels from qRT-PCR (orange), and the right vertical axis represents the FPKM values obtained from RNA-seq (green).
Fig 7.
CmWD40-mediated Alta1-induced disease resistance relies on MYC2 function.
(A) Expression of CmMYC2 in the WT and OX-CmWD40, amiR-CmMYC2, and OX-CmWD40/myc2 transgenic lines. Data are presented as the mean ± standard error of three biological replicates. Different letters at the top of error bars indicate significant differences (P <0.05, Tukey’s test). (B) Mean lesion size at 48 hpi on the WT, OX-CmWD40, amiR-CmMYC2, and OX-CmWD40/myc2 lines after pretreatment with mock solution or 300 nM Alta1 and inoculation with A. alternata. Data are presented as the mean ± standard error of four biological replicates. * P ≤0.05 compared with control, as calculated by one-way analysis of variance (ANOVA). (C) Disease symptoms caused by Alternaria alternata infection in chrysanthemum leaves of the WT, overexpression (OX)-CmWD40, amiR-CmMYC2, and OX-CmWD40/myc2 lines after pretreatment with buffer (mock) or 300 nM Alta1. Images were captured at 48 hpi; scale bar=1 cm. (D) Proposed model of AaAlta1 function during A. alternata–chrysanthemum interactions. AaAlta1 is recognized by CmWD40, which induces JA signaling to contribute to plant disease resistance.