Dissecting in vivo responses of phytohormones to Alternaria solani infection reveals orchestration of JA- and ABA-mediated antifungal defenses in potato

© The Author(s) 2022. Published by Oxford University Press on behalf of Nanjing Agricultural University. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Horticulture Research, 2022, 9: uhac188


Dear Editor,
Phytohormones play vital roles in plant survival under incessant abiotic and biotic stresses. On perception of a pathogen invasion, plants quickly activate a complex network of phytohormone signals to defend against it [1]. The general opinion is that salicylic acid (SA) mediates plant resistance to biotrophic and hemi-biotrophic pathogens and jasmonic acid (JA) acts against necrotroph [2,3]. A recent report showed that SA but not JA signaling is necessary for potato defense against Alternaria solani (A. solani), a necrotrophic pathogen causing leaf chlorosis and tissue necrosis [4]. This suggested that JA is ineffective during A. solani infection. In addition to SA, crosstalk between JA and other phytohormones has been reported widely. Thus, the function of the dialogue between JA and other phytohormones in response to necrotroph infection should be re-evaluated in potato plants. However, there is a lack of understanding about the in vivo responses of JA and other phytohormones. Here, we report the antagonistic roles of JA and abscisic acid (ABA) in response to A. solani infection in potato plants.
We inoculated potato plants (cv. Favorita, a variety susceptible to A. solani) with A. solani in a greenhouse by foliar sprays with spore suspensions (1 × 10 5 /mL) of A. solani isolate HWC-168 [5] and found slight early blight symptoms appearing occasionally on the infected leaves at 24 hours post inoculation (hpi) (Fig. 1A, 2 nd panel). The disease symptoms developed rapidly in the subsequent 24 h, and a dozen necrotic spots were observed on leaves at 36 hpi (Fig. 1A, 3 rd panel), and severe necrotic spots appeared on leaves at 48 hpi (Fig. 1A, 4 th panel). To quantify the chlorosis degree of leaves, we measured the contents of chlorophylls (chlorophyll a and b) by using leaves post 24, 36, and 48 hpi. The chlorophyll contents in the infected leaves decreased persistently at 36 hpi and 48 hpi (Fig. 1B). Based on these symptoms and chlorophyll levels, we used 24, 36, and 48 hpi as representative time points of early-, medium-, and late-infection stages, during which the antagonistic responses were classed as resistant, hindered, and failed, respectively.
Mock-inoculated (Mock) and A. solani-inoculated potato leaves at 24, 36, and 48 hpi were used for plant hormone measurement. Compared with mock samples, the levels of bioactive jasmonoyl-isoleucine (JA-Ile) were 6.7 times at 24 hpi but 0.2 times and 0.3 times at 36 hpi and 48 hpi, respectively (Fig. 1C). We noted that the JA-Ile level increased dramatically in mock at 36 hpi compared with those in other mock samples (Fig. 1C), possibly due to the expression levels of JA biosynthesis genes are higher in the daytime than at night (Fig. 1D). This result is consistent with the regulation of the plant immune response by the circadian clock in Arabidopsis [6]. A significant decrease in ABA content was triggered by A. solani infection while neither SA nor gibberellins (GAs) contents were changed markedly at three infection time points (Fig. 1C). To clarify the roles of JA and ABA in resisting A. solani, we treated potato leaves with exogenous methyl jasmonate (MeJA) or ABA, respectively, and then inoculated with A. solani. The lesions and A. solani biomasses of MeJA-or ABA-treated leaves were much smaller and less than in control treatment leaves (Fig. 1E), revealing the effects of JA and ABA on the resistance of potato to A. solani in potato.
To identify the reasons of decrease of ABA contents in potato leaves infected by A. solani, we performed mixed treatments of MeJA, ABA, and the ABA biosynthesis inhibitor (Fluridone) to the surface of potato leaves, Bars with different letters indicate significant differences between groups (p < 0.01, by Welch's ANOVA followed by the Games-Howell test), and error bars indicate 95% confidence interval. FW, fresh weight. C. Plant hormone levels in potato leaves of mock-inoculated (Mock) and A. solani-infected (A. solani) plants. Student's t test: * p < 0.05, * * p < 0.01 and * * * p < 0.001; ns represents no significant. Bars represent standard errors. Each treatment was conducted with three biological repeats. The sampling times and temperatures at 24, 36, and 48 hpi were 21:00/20 • C, 9:00/22 • C, and 21:00/20 • C, respectively. D. qPCR was used to verify the expression of eight genes responsible for JA biosynthesis. The samples were collected at 9:00/20 • C (daytime) and 21:00/22 • C (night); Student's t test: * p < 0.05, * * p < 0.01 and * * * p < 0. solani. The heat map shows the changes in gene expression. FC, fold change. K. KEGG analysis of "salmon" module genes related to phytohormone content levels. L. Phytohormone-related "samonl" module, the enlarged circle represents the hub gene encoded according to its biological function, and correlations between the hub genes are indicated by red connecting lines. WRKY33, CNGC20, RIN4, ACS6, and CALM are plant-pathogen interaction related genes. PP2C, SnRK2, ERS, TIR1, JAR1, ABF, and NPR1 are phytohormone transduction related genes. All the gene IDs in this study were listed in Table 1.

Gene Name
Gene ID Gene Name Gene ID Gene Name Gene ID Gene Name Gene ID  and then tested the levels of reactive oxygen species (ROS) by nitro blue tetrazolium (NBT) staining, and estimated the early blight disease symptoms of each sample. Through analyzing the results of NBT staining and resistance identification, we surprisingly found that the combined MeJA/ABA treatment induced ROS accumulation but did not enhance the resistant activities of potato leaves compared with control treatment, and the responses were less than MeJA treatment alone (Fig. 1F, G). Unexpectedly, the ROS accumulation and the immune activities were more intense with combined MeJA/f luridone treatment than with MeJA or f luridone treatments (Fig. 1F, 1G). Quantitative realtime PCR (qPCR) analysis showed that the transcript levels of two StMYC2 genes, responsible for JA signal transduction, were significantly lower following ABA treatment or MeJA/ABA co-treatment than in control or JA treatments, respectively (Fig. 1H). Thus, potato plants enhanced JA-mediated antifungal activity by repressing ABA contents at an early A. solani infection stage.

Figure 1D
To uncover the mechanism of hormonal reaction to A. solani infection, samples used for hormonal analysis were also used for RNA sequencing (RNA-seq). Principal component analysis (PCA) of the 36 RNA-seq datasets showed that the samples were clearly separated based on their time-course infection stage (Fig. 1I). We first focused our attention on differentially expressed genes (DEGs) involving ABA biosynthesis. As shown in Figure 1J, most genes of this pathway were reduced to different degrees at all three infestation time points of A. solani, which could have caused the decrease in ABA content. Many genes in the ABA signaling pathway (StSnRK2s) as well as ABA receptor genes (StPYLs) were induced, and StSnRK2s inhibitor coding genes (StPP2Cs) were reduced to different degrees after A. solani infection (Fig. 1J). As the ABA signaling genes could reflect feedback regulated by ABA responsive factors [7,8], we surmised that the changes in expression levels of genes in ABA signaling pathway under A. solani invasion might be a feedback response to ABA insufficiency. Consistent with this hypothesis, exogenous ABA treatment caused opposite trend of expression of genes in the ABA signaling pathway to A. solani invasion compared with control treatments, i.e. StSnRK2.6/2.7 and StPYL4 were repressed and StPP2C-24 was induced by ABA treatment (Fig. 1H). Next, we used phytohormone content as the basis to perform weighted gene co-expression network analysis (WGCNA) on the transcriptome data. After removing genes with low expression and little overall change, 6323 DEGs were selected for WGCNA. In all, 14 gene expression modules were generated in the analysis, among which DEGs identified by the salmon workflow (module) were significantly related to plant hormones. Further analysis in the kyoto encyclopedia of genes and genomes (KEGG) database revealed that DEGs in the salmon module were enriched in four types of biological activities, including protein processing, mitogen-activated protein kinase (MAPK) signaling, plant hormone signal transduction and plant-pathogen interaction (Fig. 1K). Among the DEGs of the salmon module, genes involved in plant-pathogen interaction and hormone signal transduction appeared as hubs in the network (Fig. 1L). In particular, StCNGC20, encoding a membrane channel protein that controls Ca 2+ influx [9], was associated with many genes in the module, suggesting that it might play important regulatory roles in the phytohormone-mediated plant immune response (Fig. 1L) [10].
Individual MeJA or ABA treatment enhanced plant resistance, but the activities of MeJA/ABA co-treatment were less efficient than of MeJA or ABA single treatments (Fig. 1F, G), so the plants activated only one of the two hormone pathways against A. solani. The effect of JA was better than ABA (Fig. 1E, right panel); moreover, inhibiting ABA biosynthesis enhanced the antifungal activities of JA (Fig. 1F, G), when encountering A. solani. Thus, the "best choice" for potato plants is to increase JA and decrease ABA contents. This work further our understanding of the complex coordination between JA and ABA in responding to pathogen infections in vivo and offers opportunities to discover novel strategies for improving plant immune activities.