miR825 and miR825* expression was suppressed in AR156-induced ISR to B. cinerea in Arabidopsis.
To decipher the function of miR825 and miR825* in AR156-triggered ISR to B. cinerea B1301 in Arabidopsis, wild-type Col-0 plants were pretreated with AR156 or 0.85% NaCl as a root drench and then inoculated with B. cinerea B1301 or water (mock) at 7 days post-treatment (dpt). As shown in Fig. 1a, at 2 days post-inoculation (dpi), non-treated control plants (Control/Mock) and AR156-treated plants (AR156/Mock), both of which were mock-inoculated, showed no symptoms; by contrast, with B. cinerea B1301 inoculation, both of non-treated control plants (Control/B. cinerea) and AR156-treated plants (AR156/B. cinerea) developed typical symptoms of gray mould disease (severe necrotic lesions surrounding inoculating loci, leaf yellowing, and watersoaked spots covered with B. cinerea B1301 spores), while the symptoms were less pronounced in AR156/B. cinerea than in Control/B. cinerea, indicating that AR156 pretreatment enhanced plant resistance to B. cinerea B1301. To investigate whether miR825 and miR825* have function in the mediation of AR156-triggered ISR against B. cinerea B1301, we reasoned that if having this function, they may be differentially expressed during the process of priming or upon B. cinerea B1301 inoculation. Therefore, we examined the expression of the two miRNAs in AR156/B. cinerea, Control/B. cinerea, AR156/Mock, and Control/Mock at 48 hours post-inoculation (hpi). Northern blotting analysis confirmed that miR825* expression was downregulated with B. cinerea B1301 infection in both AR156-treated and control plants. On the one hand, there was no appreciable difference in the level of miR825* expression between AR156/Mock and Control/Mock, yet its level was significantly lower in AR156/B. cinerea than in Control/B. cinerea, and the difference in the same parameter between Control/Mock and Control/B. cinerea was smaller than that between AR156/Mock and AR156/B. cinerea (Fig. 1b). In addition, we also investigated the role of miR825 in ISR. On the one hand, no difference in miR825 expression level was noted between AR156/Mock and Control/Mock, which was similar to the expression profile of miR825*; on the other hand, though miR825 expression was also downregulated upon B. cinerea B1301 infection in control plants, the downregulation was significantly stronger in AR156-treated plants (Fig. 1b). The results indicated that AR156 pretreatment resulted in significant suppression of the expression of miR825 and miR825*, suggesting that they might function as negative regulators of AR156-mediated ISR to B. cinerea B1301.
miR825 and miR825* participate in AR156-induced ISR.
To further investigate whether miR825 and miR825* function in AR156-elicited ISR to B. cinerea B1301, the ability of AR156 to trigger ISR was evaluated in the transgenic plants in which miR825 and miR825* were knockdowned by using STTM strategy and in miR825 and miR825* OE plants. Two-way ANOVA with Tukey’s test was used to compare the levels of resistance to B. cinerea B1301 in AR156-treated and non-treated plants with different genotypes. Typical symptoms of gray mould disease (severe necrosis and watersoaked spots surrounded by the spores) appeared on leaves of Control/B. cinerea from Col-0 at 2 dpi (Fig. 2a); in comparison, disease symptoms were more severe in those from miR825/825* OE lines (#50 and #56), which formed larger necrotic lesions, but lighter in those from STTM825/825* transgenic lines (#1 and #3) (Fig. 2a). However, AR156 pretreatment led to marked reductions in disease symptoms regardless of plant lines (Fig. 2a). On the other side, compared with Col-0, STTM825/825* lines (#1 and #3) expressed stronger resistance to Phytophthora capsici (P. capsici) (Additional file 1: Figure S1A) and cucumber mosaic virus (CMV) (Additional file 1: Figure S1B), and showed significant (**P < 0.01) reductions in the lesion diameter on the leaf infected by P. capsici (Additional file 1: Figure S1C) and in that by CMV (Additional file 1: Figure S1D), indicating that miR825 and miR825* act as negative regulators of Arabidopsis resistance to various pathogens (B. cinerea, P. capsici, and CMV). Compared to Col-0 plants, the diameter of leaf necrosis was smaller in the two STTM825/825* lines, but bigger in the two miR825/825* OE lines, and AR156-treated plants from STTM825/825* lines developed significantly (**P < 0.01) smaller necrotic lesions on leaves than the corresponding control plants (Fig. 2b). Additionally, qRT-PCR was conducted to measure B. cinerea B1301 growth rate at 2 dpi in infected leaves. Coincident with the reduction in disease symptoms on AR156-treated leaves, the growth rate of B. cinerea B1301 was consistently lower in leaf tissues of AR156-pretreated plants with three different genotypes than in respective control plants; with AR156 pretreatment, the fungal growth rate was lower in the STTM825/825* lines, but higher in the miR825/825* OE lines than in Col-0 plants (Fig. 2c). The two-way ANOVA revealed that the interaction between treatment (AR156, Control) and genotype was statistically significant and that AR156-pretreated plants with three different genotypes overall showed significantly higher levels of resistance to B. cinerea B1301 than respective control plants. Collectively, these results implicate that miR825 and miR825* function to suppress innate immunity in Arabidopsis that protect plants from the infection of a wide variety of pathogens (B. cinerea, P. capsici, and CMV).
miR825 and miR825* affect defense-related gene expression and cellular defense responses in AR156-primed plants upon pathogen attack.
To identify the signaling pathway(s) involved in AR156-induced ISR, qRT-PCR was used to examine expression levels of the reporter genes of the SA-signaling pathway (PR1, PR2, and PR5) and that of the JA/ET-signaling pathway (PDF1.2). The expression of these genes in plants with different genotypes was analysed with two-way ANOVA. Without B. cinerea B1301 infection, their expression levels in the miR825/825* OE lines (#50 and #56) and STTM825/825* transgenic lines (#1 and #3) were comparable to those in the wild-type plants at 48 hpi (Fig. 3a). However, their transcription was significantly enhanced in AR156/B. cinerea relative to that in Control/B. cinerea at 48 hpi regardless of plant lines (Fig. 3a). Moreover, the two-way ANOVA revealed that the interaction between treatment effect and genotype effect was statistically significant; with AR156 pretreatment, the transcriptional levels of these genes significantly increased in the STTM825/825* transgenic plants, but declined in the miR825/825* OE plants compared with those in Col-0 plants (Fig. 3a), which conformed to the function of miR825/825* as negative regulators of AR156-mediated ISR to B. cinerea.
We also investigated whether miR825 and miR825* act in AR156 priming the plant for potentiated cellular defense responses, represented by the accumulation of hydrogen peroxide and the deposition of callose, in Arabidopsis upon pathogen attack. As shown in Fig. 3b, the two events were evident at 12 hpi in AR156/B. cinerea of both Col-0 and STTM825/825* transgenic plants (lines 1 and 3), but much weaker in that of miR825/825* OE plants (lines 50 and 56); meanwhile, they were absent in Control/B. cinerea. With AR156 pretreatment, these defense responses were potentiated in Col-0 and the two STTM825/825* transgenic lines at 24 hpi; meanwhile, they were weaker in both Control/B. cinerea and AR156/B. cinerea of miR825/825* OE plants (Fig. 3b). The observed results indicated that upon pathogen attack, AR156-treated Arabidopsis plants expressed faster and stronger cellular defense responses, which, however, could be dampened by miR825 and miR825*.
miR825 and miR825* participate in regulation of PTI components in AR156-elicited ISR.
With the purpose of elucidating the role of miR825 and miR825* in AR156-triggered ISR and PTI, the phosphorylation of MPK3 and MPK6 was examined in both control and AR156-pretreated plants inoculated with B. cinerea B1301. Using antibodies specifically recognizing MPK3 and MPK6, their phosphorylation was detected in leaves of Control/B. cinerea of Col-0 at 10 min post-inoculation (mpi); then its intensity gradually declined at 30 and 60 mpi. However, AR156 pretreatment caused sustained phosphorylation of the two kinases in leaves of Col-0 plants; its intensity increased with time, peaking at 60 mpi (Fig. 4a). In Control/B. cinerea and AR156/B. cinerea from miR825/825* OE line (#50), the tendency of MPK3 and MPK6 phosphorylation was the same as that in the corresponding treatments from Col-0 (Fig. 4b). Notably, the phosphorylation of the two kinases was sustained from 10 to 60 mpi in both Control/B. cinerea and AR156/B. cinerea of miR825/825* knockdown line (#1), with its intensity remaining substantially higher in AR156/B. cinerea than in Control/B. cinerea over the time course (Fig. 4c). These results suggested that AR156-triggered ISR involves an enhanced and sustained PTI response, in which miR825 and miR825* play a negative regulatory role.
To search further evidence to support our hypothesis, we evaluated the transcription levels of two PTI marker genes: the flg22-induced receptor-like kinase 1 (FRK1) and WRKY53 [43, 44]. Differences in their expression between AR156-pretreated and control plants with different genotypes upon B. cinerea B1301 infection were analysed by two-way ANOVA. The qRT-PCR assay revealed that the expression of FRK1 and WRKY53 was enhanced and sustained in control/B. cinerea of Col-0, miR825/825*OE line (#50), and STTM825/825* transgenic line (#1) from 10 to 60 mpi. However, their expression significantly increased at 30 and 60 mpi in the AR156/B. cinerea of STTM825/825* lines relative to that in the corresponding control plants (Fig. 4d). The two-way ANOVA revealed a significant (P < 0.01) interactive effect of treatment and genotype. These results confirmed that miR825 and miR825* negatively regulate the expression of PTI components in AR156-mediated ISR.
Target genes of miR825 and miR825* are expressed in a similar manner in AR156-induced ISR.
To investigate whether miR825 and miR825* modulate ISR by affecting the expression of their targets genes, qRT-PCR was conducted to examine expression patterns of miR825/825* target genes in control and AR156-pretreated plants of Col-0, miR825/825* OE lines (#50 and #56), and STTM825/825* lines (#1 and #3) with or without B. cinerea B1301 inoculation. We predicted that miR825/825* targets and their cognate miRNAs would exhibit opposite expression patterns in ISR-expressing plants. The expression of the targeted genes in plants with different genotypes treated with or without AR156 was analyzed using two-way ANOVA. As expected, the results of qRT-PCR indicated that the transcriptional levels of three miR825*-targeted genes (AT5G40910, AT5G38850, and AT3G04220) and a miR825-targeted gene (AT5G44940) were all significantly improved in leaves of both control and AR156-pretreated plants from miR825/825* knockdown lines (#1 and #3) with or without B. cinerea B1301 inoculation compared with those from Col-0 at 48 hpi. In contrast, their expression levels were consistently reduced in four treatments (Control/Mock, AR156/Mock, Control/B. cinerea, and AR156/B. cinerea) from miR825/825* OE lines (#50 and #56) compared with Col-0 at the same time. Moreover, AR156-induced transcription of the four target genes was significantly stimulated in the STTM825/825* transgenic plants, but attenuated in the miR825/825* OE plants relative to that in Col-0 plants (Fig. 5a). At the same time, AR156 pretreatment coupled with B. cinerea B1301 infection led to conspicuous up- and down-regulation of these genes in STTM825/825* and miR825/825* OE plants, respectively, compared with that in Col-0 plants (Fig. 5a). Two-way ANOVA revealed that the interaction between treatment effect and genotype effect was statistically significant. Hence, it is clear that miR825 and mi825* function to suppress AR156-elicited ISR.
To further confirm that these genes are targeted by miR825 and miR825*, the two miRNAs were separately transiently co-expressed with a target gene fused with the HA-tag in Nicotiana benthamiana. An anti-HA antibody was used to detect the proteins encoded by AT5G40910, AT5G38850, AT3G04220, and AT5G44940. As a result, their expression was apparently repressed by the co-expression of miR825/825*, but not by miR319b, which is irrelevant to these genes (Fig. 5b). Taken together, these results signified that the four genes are authentic miR825- or miR825*-targeted genes participating in AR156-induced ISR against B. cinerea B1301.
Plants silencing miR825/miR825*-targeted genes are more susceptible to B. cinerea.
To clarify the function of the target genes of miR825 and miR825*, they were evaluated in a functional analysis by using the null mutants at5g40910 (SALK_043422C), at5g38850 (SALK_134889C), at3g04220 (CS384498), and at5g44940 (SALK_021558C), each with a T-DNA insertion in an intron or exon (Additional file 2: Figure S2A). Semi-quantitative RT-PCR confirmed that the two mutants At5g38850 and At3g04220 were homozygous mutants (Additional file 2: Figure S2B); however, this assay also indicated that we failed to obtain the homozygous mutants of AT5G40910 and AT5G44940 (Additional file 3: Figure S3), and we speculate that this failure might be caused by homozygous infertility or T-DNA insertion mutation. The germination rate of seeds was similar among Col-0, at5g38850, and at3g04220 (Additional file 2: Figure S2C). The mutants at5g38850 and at3g04220 phenotypically developed to a lesser degree than Col-0 plants (Additional file 2: Figure S2D). To elucidate the role of these target genes in AR156-induced ISR to B. cinerea B1301 in Arabidopsis, we assessed disease development in Col-0 and the two mutant lines at 2 dpi. Two-way ANOVA with Tukey’s test was conducted to compare the differences in disease severity between AR156-treated and non-treated plants with different genotypes infected with the pathogen. As shown in Fig. 6a, B. cinerea B1301 infection caused severe disease symptoms on leaves of control plants from both Col-0 and the two mutant lines, while the symptoms were more severe in the mutants than in Col-0, indicating the mutant lines were more prone to the attack by B. cinerea B1301 than Col-0 (Fig. 6a). However, AR156 pretreatment caused a noticeable attenuation of disease symptoms on plants of Col-0 and the two mutants, demonstrating that AR156 induced effective ISR in both Col-0 and the mutants. Consistently, AR156-treated plants of Col-0 and target mutant lines developed significantly (**P < 0.01) smaller necrotic lesions on leaves than their respective control plants; on the other hand, the lesions in the target mutants were larger than those in Col-0 plants (Fig. 6b). The fungal growth rates in plants of Col-0 and the two target mutant lines were also determined by qRT-PCR. In consistence with the inhibitory effect of AR156 pretreatment on the disease symptoms observed on leaf surface, the growth of B. cinerea B1301 at 2 dpi was significantly slower in AR156/B. cinerea than in Control/B. cinerea regardless of plant lines; on the other hand, the fungal growth rate increased in target mutants than in Col-0 plants regardless of pretreatment (AR156 or Control) (Fig. 6c). Importantly, the two-way ANOVA showed a significant interactive effect of treatment and genotype, as well as a significant treatment effect demonstrated by that the overall levels of resistance to B. cinerea B1301 in AR156-pretreated plants significantly exceeded those in control plants across different genotypes. Taken together, the target mutant plants showed increased susceptibility to the pathogen, yet AR156 still retained the capacity to trigger ISR in them.