The m6A reader MhYTP2 negatively modulates apple Glomerella leaf spot resistance by binding to and degrading MdRGA2L mRNA

Abstract Glomerella leaf spot (GLS), caused by the fungal pathogen Colletotrichum fructicola, significantly threatens apple production. Some resistances to plant disease are mediated by the accumulation of nucleotide‐binding site and leucine‐rich repeat (NBS‐LRR) proteins that are encoded by a major class of plant disease resistance genes (R genes). However, the R genes that confer resistance to GLS in apple remain largely unclear. Malus hupehensis YT521‐B homology domain‐containing protein 2 (MhYTP2) was identified as an N 6‐methyladenosine RNA methylation (m6A) modified RNA reader in our previous study. However, whether MhYTP2 binds to mRNAs without m6A RNA modifications remains unknown. In this study, we discovered that MhYTP2 exerts both m6A‐dependent and ‐independent functions by analysing previously obtained RNA immunoprecipitation sequencing results. The overexpression of MhYTP2 significantly reduced the resistance of apple to GLS and down‐regulated the transcript levels of some R genes whose transcripts do not contain m6A modifications. Further analysis indicated that MhYTP2 binds to and reduces the stability of MdRGA2L mRNA. MdRGA2L positively regulates resistance to GLS by activating salicylic acid signalling. Our findings revealed that MhYTP2 plays an essential role in the regulation of resistance to GLS and identified a promising R gene, MdRGA2L, for use in developing apple cultivars with GLS resistance.

affecting fruit production the following year (Velho et al., 2015).The apple cultivars Golden Delicious, Gala, and Qinguan are highly susceptible to GLS (Liu et al., 2016).
So far, research on GLS in apple has focused on the isolation and identification of the pathogen (Moreira et al., 2019), identifying environmental conditions that affect its infection and spread (Wang et al., 2015), and determining control methods (Zhang et al., 2016).
However, because there is no effective approach that is currently used to control GLS, our inability to protect susceptible apple varieties from C. fructicola infection results in significant loss of apple production (Wang et al., 2015).Therefore, clarifying the molecular mechanisms of disease resistance and breeding apple cultivars that are resistant to C. fructicola have become major objectives of China's apple breeding programme.
Plants have evolved complex mechanisms to induce defence reactions (Dangl & Jones, 2001;Ramirez-Prado et al., 2018;Sharifi et al., 2018).Plant defence responses are mediated by pattern recognition receptors (PRRs) and cytoplasmic immune receptors (van Esse et al., 2020).The recognition of highly conserved pathogenassociated molecular patterns (PAMP) by host cell surface PRRs is called pattern-triggered immunity (PTI) (Parker et al., 2022).PTI triggers cytological and physiological responses that enable plants to resist pathogen infection, such as responses in nitric oxide, hormones, and expression of disease-resistance genes (Xu et al., 2022;Yu et al., 2017).Use of the cytoplasmic immune receptors is a wellknown strategy that is characterized by specific interactions between a plant's resistance (R) gene and a pathogen's corresponding avirulence (Avr) gene leading to disease resistance (Ji et al., 2021).
The largest group of R genes contain one or more nucleotide-binding sites and leucine-rich repeat (NBS-LRR) domains that recognize virulence effectors, thus leading to effector-triggered immunity (ETI) (Arya et al., 2014;McHale et al., 2006;Steinbrenner et al., 2015;Teixeira et al., 2014;Zhong et al., 2015).Plant disease resistance is characterized by the accumulation of NBS-LRR proteins that are encoded by R genes.PTI and ETI, two major immune pathways in plants, amplify each other and act synergistically to ensure that plants can produce a lasting and strong immune response to pathogen invasion (Ngou et al., 2021;Yuan et al., 2021).In the ETI response, R genes can activate signalling pathways downstream of salicylic acid (SA) (Chen et al., 2021).SA plays an important role in defending plants against biotrophic pathogens (Samaradivakara et al., 2022).Additionally, SA levels in plants are mainly determined by the balance between SA biosynthesis and SA degradation.SA biosynthesis is mainly controlled by isochorismate synthase 1 (ICS1) (Seyfferth & Tsuda, 2014).SA hydroxylation is the main pathway mediating the degradation of SA.SA can be hydroxylated by the enzyme encoded by Downy Mildew Resistant 6 (DMR6; Zhang et al., 2013Zhang et al., , 2017)).In apple (Malus domestica), MdDMR6 is a homologue of AtDMR6 and encodes SA hydroxylase, which converts SA into 2,5-dihydroxybenzoic acid, resulting in the inactivation of SA (Shan et al., 2021;Zhang et al., 2017).
Studies have also shown that MhYTP2 functions in basic RNA stability and powdery mildew (Podosphaera leucotricha) resistance (Guo et al., 2022).In the present study, we identified that MhYTP2 was also involved in apple resistance to GLS by directly binding to the mRNA of MdRGA2L, which encodes a coiled-coil (CC)-NBS-LRR protein, leading to the down-regulation of MdRGA2L transcripts on C. fructicola infection.MdRGA2L enhanced resistance to GLS by activating SA signalling in apple.The identification of MdRGA2L provides a new in-depth understanding of a molecular mechanism that underlies immunity to GLS in apple and could promote the molecular breeding of GLS-resistant apple varieties.

| MhYTP2 reduces the resistance to GLS
We occasionally found that three 35S::MhYTP2 overexpression lines (OE-1, OE-2, OE-3) showed reduced resistance to GLS compared with the wild-type (WT) plants under field conditions.To verify this discovery, mature leaves from the three 35S::MhYTP2 lines and the WT apple trees were tested for their resistance to GLS in the laboratory.At 6 days postinoculation (dpi), the 35S::MhYTP2 lines were more severely infected than the WT plants (Figure 1a,b).SA plays an important role in plant disease resistance.As shown in Figure 1c, inoculation with C. fructicola caused SA to accumulate in all the lines at 6 dpi compared with the group that was not inoculated.The SA content had no significant differences among the WT and 35S::MhYTP2 plants in the absence of C. fructicola inoculation.However, after 6 days of C. fructicola infection, the SA content was significantly reduced in the 35S::MhYTP2 lines compared with the WT plants (Figure 1c).The SA biosynthetic gene ICS1 was consistently associated with SA content.The pathogenesis-related 1 (PR1) gene exhibited no significant differences between the WT and 35S::MhYTP2 plants in the absence of C. fructicola infection.At 6 dpi, the level of expression of MdPR1 in the 35S::MhYTP2 lines was significantly lower than that in the WT plants (Figure 1d).These results suggest that the decreased resistance of 35S:MhYTP2 apple lines to GLS could be related to the decrease in SA content.

| MhYTP2 binds to a portion of the target mRNAs that is not m 6 A modified, including the mRNAs of R genes
We analysed the results of a previous RNA immunoprecipitation sequencing (RIP-Seq) analysis (Guo et al., 2022).A Gene Ontology (GO) enrichment analysis of MhYTP2-bound transcripts without N 6 -methyladenosine RNA methylation (m 6 A) modification was performed to gain functional insights into the role of MhYTP2 independent of m 6 A. The analysis showed that the MhYTP2-bound transcripts without m 6 A modification were enriched in multiple signalling pathways and cellular processes (Figure 2).Because MhYTP2 negatively regulates GLS resistance, we focused on the genes that were enriched in the defence response pathways.Further analysis showed that MhYTP2 was bound to the mRNAs of 58 R genes that encode proteins with NBS-LRR domains (Table S1).

| MhYTP2 binds to MdRGA2L mRNA and reduces its stability
MdRGA2L (MD07G1016200) is one of the 58 R genes bound by MhYTP2 that was enriched in defence response pathways, and it had the most numerous RIP-Seq reads (Figure 3a).R genes play crucial roles in the regulation of plant resistance to biotic stress (Lee & Lee, 2005;Nandety et al., 2013).Thus, MdRGA2L was selected for further study.An electrophoretic mobility shift assay (EMSA) showed MhYTP2 binding to MdRGA2L mRNA (Figure 3b).Studies have demonstrated that MhYTP2 binds to and affects the stability of mRNA (Guo et al., 2022).We hypothesized that the role that MhYTP2 plays in the stability of MdRGA2L mRNA could affect the defence of apple against GLS, therefore the degradation rate of MdRGA2L was measured.The MdRGA2L transcript was rapidly degraded in the 35S::MhYTP2 line OE-2 compared with the WT plants (Figure 3c), whereas the mRNA lifetimes of the negative control gene MdMDH in the 35S::MhYTP2 line OE-2 and the WT plant were similar (Figure S1).This suggested that MhYTP2 promoted the degradation of MdRGA2L mRNA.

| MdRGA2L is an R gene induced by C. fructicola and localized to the nucleus and cytosol
A phylogenetic analysis revealed that MdRGA2L shares high similarity with Chinese white pear (Pyrus bretschneideri) RGA2L (Figure 4a).
We examined the changes in the transcript level of MdRGA2L between the GLS-susceptible cultivar Gala and the GLS-resistant cultivar Fuji under both control and C. fructicola inoculation conditions.As shown in Figure 4b, the transcript levels of MdRGA2L did not change significantly in the leaves of the uninoculated plants.On infection, the level of transcripts increased 6.3-and 1.3-fold on the fifth day following inoculation with C. fructicola relative to that on the fifth day under the control conditions in Fuji and Gala cultivars, F I G U R E 2 Gene Ontology (GO) enrichment analysis of the MhYTP2-bound transcripts that were not modified by N 6 -methyladenosine RNA methylation.respectively (Table S2).This result suggests that MdRGA2L might be involved in apple resistance to C. fructicola.MdRGA2L was observed to be localized to the nucleus and cytosol (Figure 4c).

| MhYTP2 decreased the transcript levels of MdRGA2L on C. fructicola infection
We monitored the levels of expression of MdRGA2L in the WT and 35S::MhYTP2 plants.The transcript levels of MdRGA2L had no clear differences between the WT and 35S::MhYTP2 plants under control growth conditions.After inoculation with C. fructicola for 6 days, the transcript levels of MdRGA2L were up-regulated in both the WT and 35S::MhYTP2 plants, but the 35S::MhYTP2 lines had an evident reduction compared with the WT plants (Figure 5a).MhYTP2 was previously shown to affect the translation efficiency of the bound mRNAs (Guo et al., 2022).To explore whether MhYTP2 affects the translation efficiency of the 58 R genes, we referred to the ribosome profiling (Ribo-Seq) results of Guo et al. (2022), and 19 genes that had significant changes (fold change ≥2; p < 0.05) were found between the 35S::MhYTP2 lines and WT plants (Figure 5b).Among these 19 R genes, the translation efficiency of 14 significantly decreased and five increased.These data indicate that MhYTP2 is involved in the regulation of the translation efficiency of R genes.
We did not observe a significant change in the translation efficiency of MdRGA2L between the WT and 35S::MhYTP2 plants.
We monitored the levels of MdRGA2L protein in comparison with the performance of WT plants.The MdRGA2L protein significantly

| MdRGA2L enhanced apple GLS resistance by activating SA signalling
To explore the role of MdRGA2L in defending against GLS, its expression was modified by transforming an overexpression construct (MdRGA2L-OE) or an antisense suppression construct (MdRGA2L-Ri) into leaves of the M. domestica genotype GL-3 (cv.Royal Gala) (Figure S2).When these 2-day-old empty vector (EV) and transgenic apple leaves were infected with C. fructicola for 3 days, the MdRGA2L-OE apple leaves were strongly resistant to GLS compared with the EV control.In contrast, the MdRGA2L-Ri apple leaves showed the opposite phenotype (Figure 6a).We also found that the MdRGA2L-OE plants were more strongly resistant to GLS than the others when treated with C. fructicola for 6 days (Figure S3).To further confirm the role of MdRGA2L in the process of plant GLS resistance, we knocked down MdRGA2L in the young leaves of Fuji, a variety that is resistant to GLS (Figure S4).The leaves were inoculated with C. fructicola 2 days later, and we found that Fuji was no longer resistant to GLS (Figure 6b).These observations indicate that MdRGA2L has a substantial role in the defence of Fuji plants against GLS.In this experiment, we also observed that the contents These results suggest that MdRGA2L may execute its role in GLS tolerance by regulating the contents of SA.To test this, MdRGA2L and the SA degradation gene MdDMR6 were co-expressed in apple leaves.When challenged with C. fructicola for 3 days, the apple leaves that overexpressed MdRGA2L and MdDRM6 showed the same resistance to GLS as the EV control (Figure 6a).Moreover, to further determine whether MdRGA2L is involved in the SA signalling pathway, MdRGA2L-Ri apple leaves (2 days after infiltration) were treated with 200 mg/L SA before they were inoculated with C. fructicola, MdRGA2L-Ri apple leaves showed a similar phenotype as the EV control (Figure 6a).Collectively, these results indicate that MdRGA2L enhances the resistance of apple to GLS by activating SA signalling.Surprisingly, we found that the whole plant exhibited resistance to GLS, including the leaves in which MdRGA2L was transiently overexpressed (infiltrated leaves) and the upper leaves and lower leaves that lacked transient MdRGA2L overexpression.Thus, we hypothesized that SA, which plays a critical role in systemic acquired resistance, was transported from the infiltrated leaves to the other leaves.To confirm this, we separately collected the infiltrated leaves in which MdRGA2L was transiently overexpressed and the upper and lower uninfiltrated leaves and measured their SA contents.The results showed that the levels of SA in all the samples of MdRGA2L-OE plants tested were higher than those of the EV control, whereas the levels of SA in all the samples of MdRGA2L-Ri plants tested were lower than those of the EV control (Figure 6c).These results suggest that MdRGA2L regulates the resistance of apple to GLS by promoting the biosynthesis of SA.

| MdRGA1 has no function in GLS resistance
MhYTP2-bound R genes can be primarily divided into three categories: CC-NBS-LRR proteins, NBS-LRR proteins, and toll/interleukin-1 receptor (TIR)-NBS-LRR proteins (Table S2).Among them, the CC-NBS-LRR proteins and TIR-NBS-LRR proteins were both more highly expressed when induced by C. fructicola than were the NBS-LRR proteins (Figure 7a-c).We showed that MdRGA2L (a CC-NBS-LRR protein) plays a fundamental role in the resistance of apple to GLS.However, it is not known whether another MhYTP2 target, MdRGA1 (MD07G1015300), the TIR-NBS-LRR gene that is the most significantly induced by GLS, plays a role in GLS resistance.To explore this, we overexpressed and knocked down MdRGA1 in apple leaves and found that both types of leaves showed no difference in GLS resistance compared with the control plants (Figure 7d), implying that MdRGA1 has no function in the resistance of apple plants to GLS.

| DISCUSS ION
GLS induces yield losses that have ranged from 30% to 70% over the past decade in China (Zhang et al., 2008).GLS weakens the fruit tree vitality, causes fruit necrosis, and reduces fruit quality owing to the defoliation of leaves (Shan et al., 2021;Velho et al., 2016;Wang et al., 2015).Additionally, GLS develops rapidly and is difficult to prevent (Lv et al., 2022;Shang, Wang, et al., 2020;Zhang et al., 2016).At present, there is no effective way to control GLS outbreaks.The extensive application of a variety of fungicides has not effectively controlled the disease (Wang et al., 2015).Previous studies explored the mechanisms controlling apple defence against GLS.However, the molecular mechanisms that underlie the resistance of apple to GLS remain poorly understood.
Most of the studies on the regulation of disease resistance using m 6 A readers were performed in mammals.The RNA m 6 A reader YTHDF2 targets AXIN1 and subsequently affects its stability to promote proliferation and metastasis of lung adenocarcinoma cells (Li et al., 2021).YTHDF2 promotes the degradation of mRNA and cancer progression by increasing its binding affinity to m 6 A-modified mRNAs (Hou et al., 2021).The nuclear m 6 A reader YTHDC1 plays a critical role in leukemogenesis by regulating MCM complex-mediated DNA replication (Sheng et al., 2021).The nuclear m 6 A reader IGF2BP enhances mRNA stability and translation, which enables the development of cancer (Huang et al., 2018).However, the available functional data are scarce in plants.MhYTP2 positively regulates resistance against powdery mildew by binding to and affecting the stability of MdMLO19 and MdPAL1 mRNAs in apple as an m 6 A reader (Guo et al., 2022).To our knowledge, this study is the first to report that MhYTP2, a YTHdomain-containing RNA-binding protein, negatively modulates the defence of apple against GLS by regulating the stability of MdRGA2L mRNA in an m 6 A-independent manner.NBS-LRR proteins can be divided into two classes according to their N-terminal domains, including the toll/interleukin-1 receptor (TIR) domain for NBS-LRR proteins (TNL) and the coiled-coil (CC) domain for NBS-LRR proteins (CNL) (Funk et al., 2018).In the present study, we discovered that MdRGA2L, which encodes a CNL protein, is a functional R gene in apple.Its overexpression substantially reduced the susceptibility to GLS, whereas its inactivation substantially increased the susceptibility to GLS.The overexpression of MhYTP2 accelerated the degradation of MdRGA2L mRNA, leading to a reduction in resistance against GLS.We previously demonstrated that MhYTP2 positively regulates apple resistance against drought, salt, and powdery mildew when overexpressed (Guo et al., 2022;Liu et al., 2018;Wang, Guo, Sun, Wang, Shao, Liang, et al., 2017;Wang, Guo, Wang, Sun, Shao, Liang, et al., 2017).
How can this GLS susceptibility weakness of MhYTP2 be overcome?
Considering that MhYTP2 directly binds to MdRGA2L mRNA and accelerates its degradation, the mutation of MdRGA2L at suitable sites with genome editing technology may help its mRNA avoid recognition by MhYTP2, which could be used to maintain GLS resistance.
TNLs have been reported as resistance genes in Arabidopsis thaliana (Gassmann et al., 1999), grape (Vitis vinifera) (Li et al., 2017), and rice (Oryza sativa) (Zhao et al., 2016).In apple plants, the apple scab (Venturia inaequalis) resistance locus Rvi15 (Vr2) has been found to contain three TNL genes (Galli et al., 2010).The TNL gene MdTNL1 is the key factor that regulates the resistance of apple to GLS (Lv et al., 2022).In the present study, we determined that the TNL gene MdRGA1 has no function in the resistance of apple to GLS.Some studies showed that deletion of the N-terminal domain of NBS-LRRs could not induce plant immunity.However, the expression of some N-terminal domains of NBS-LRRs alone is sufficient to elicit a hypersensitive response (Collier et al., 2011).In the present study, our findings did not reveal whether the CC domain of MdRGA2L induces plant immunity or not.In addition, whether or not the CC domain of MdRGA2L is involved in GLS resistance needs to be determined in the future.
SA plays an important role in defending plants against biotrophic pathogens (Samaradivakara et al., 2022).Exogenous application of SA can result in strong resistance to GLS (Zhang et al., 2016).Plants immediately stimulate their own PTI and ETI responses to respond when they perceive pathogens, and both of these immune responses are related to SA. PTI can promote the expression of ICS1, which leads to the gradual accumulation of SA (Wildermuth et al., 2001).In the ETI reaction, the recognition of R genes in host cells with effector proteins secreted by pathogens can activate signalling pathways downstream of SA (Chen et al., 2021).
Based on these experimental results, we summarized the model of MhYTP2 regulating plant resistance to GLS (Figure 8).In this model, C. fructicola infection up-regulates the expression of MdRGA2L.
MhYTP2 directly binds to the mRNA of MdRGA2L and decreases its stability, which reduces the expression level of MdRGA2L after inoculation with C. fructicola.MdRGA2L directly increases the SA content by promoting the expression of the SA synthesis-related gene ICS1.
Subsequently, the increase in SA induces systemic acquired resistance.
Thus, MdRGA2L has been identified to have substantial potential in breeding excellent apple varieties that are resistant to GLS.

| Plant materials
The WT and MhYTP2 transgenic M. domestica 'Royal Gala' plants were the same as those previously used in drought experiments (Liu et al., 2018).
For MdRGA2L and MdRGA1 GLS resistance assays, tissuecultured WT plants and transgenic culture conditions are as previously described (Guo et al., 2019).The tobacco (Nicotiana tabacum) plants were grown under a 16 h:8 h light:dark photoperiod at 21°C in a growth chamber.

| Fungal culture, leaf infection, and disease incidence statistics
The cultures of the pathogenic fungus C. fructicola were obtained and maintained as previously described (Li et al., 2023;Lv et al., 2022;Shang, Liang, et al., 2020), and 10 6 cfu/mL inoculum was used to inoculate the plants (Figure S5).Each inoculum was quantified by microscopy (CX31RTSF; Olympus) and used to inoculate detached apple leaves by spraying them with a spore suspension.
Mature leaves were collected from trees growing in the field and disinfected with 75% (vol/vol) ethanol, then the bottoms of their petioles were wrapped with wet cotton balls prior to culture at 75% humidity and 25°C.Six days after inoculation, the necrotic lesions of each leaf were visually observed and recorded.Three biological replicates were performed on about 30 leaves from different plants.
The expression vectors were constructed by inserting the open reading frames of MdRGA2L, MdRGA1, or MdDRM6 into the pCam-bia2300 vector.The vectors pHellsgate2 and pK7WIWG2D were used as RNAi-mediated vectors to silence MdRGA2L or MdRGA1 as described by Zhou et al. (2017).These vectors were then transferred to Agrobacterium tumefaciens EHA105 to mediate transient expression in apple leaves.The functions of MdRGA2L and MdRGA1 in apple leaves were analysed using the Agrobacterium-mediated transient expression method (Zhang et al., 2018).Three days after agroinfiltration, six apple leaves were collected from different plants to detect the relative transcription levels of target genes and those of the genes related to the defence response in apple.The experiments were conducted in triplicate.
The areas of leaves with disease symptoms and the areas covered by GLS fungus were analysed using ImageJ software (NIH).
The plant cells were visualized using propidium iodide (Sigma-Aldrich).
The sample processing and imaging observations were conducted as previously described (Guo et al., 2022).To obtain fluorescence of the fungal hyphae and plant cells, excitation wavelengths of 488 nm and F I G U R E 8 The proposed model describes how the m 6 A reader protein MhYTP2 regulates apple resistance to Glomerella leaf spot.SA, salicylic acid.

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MhYTP2 reduces resistance to Glomerella leaf spot (GLS).(a) Disease severity recorded and statistics of GLS susceptibility 6 days following inoculation with Colletotrichum fructicola of the overexpression transgenic lines (OE-1, OE-2, and OE-3) and the wild-type (WT) plants.Scale bars 1 cm.(b) The spread of the pathogen and its statistical summary in the inoculated apple leaves.Scale bars 1 mm.(c) The contents of salicylic acid (SA) in the transgenic lines and WT plants on the sixth day after inoculation with C. fructicola (6 days-Treatment) or in the absence of C. fructicola (6 days-Control).FW, fresh weight.(d) The transcript levels of MdICS1 and MdPR1 in the transgenic lines and the WT plants on the sixth day after inoculation with C. fructicola (6 days-Treatment) or in the absence of C. fructicola (6 days-Control).Data are presented as mean ± SD.Different letters indicate significant differences among the WT and transgenic plants on the same day of different treatments.

F
I G U R E 3 MhYTP2 binds to MdRGA2L (MD07G1016200) mRNA and reduces the stability of MdRGA2L mRNA.(a) Data from RNA immunoprecipitation sequencing shows that MhYTP2 binds to MdRGA2L mRNA.The ratio values on the right of the heatmap represent the ratio of the number of reads of MhYTP2-F2 to that of FLAG.FLAG represents transgenic 35S::FLAG apple callus and MhYTP2-F2 represents transgenic 35S::MhYTP2-FLAG apple callus line 2. (b) Validation of MhYTP2 binding to MdRGA2L mRNA by electrophoretic mobility shift assay.(c) The mRNA lifetime of MdRGA2L in the transgenic line OE-2 and the wild-type (WT) plants.MhYTP2 nontarget MdMDH was used as the negative control.Data are represented as the mean ± SD. decreased in 35S::MhYTP2 plants after inoculation with C. fructicola, but there was little difference among the WT and 35S::MhYTP2 plants under control growth conditions (Figure 5c).These data indicate that MhYTP2 is involved in regulating the translation efficiency of R genes.MhYTP2 regulates the expression of MdRGA2L at the posttranscriptional level but not at the translational level.

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I G U R E 4 MdRGA2L is an R gene induced by Colletotrichum fructicola and localized to the nucleus and cytosol.(a) Phylogenetic and conserved structural domain analysis of MdRGA2L in plants.(b) The changes in transcript level of MdRGA2L during the progress of inoculation with C. fructicola of the Glomerella leaf spot (GLS)-susceptible cultivar Gala and GLS-resistant cultivar Fuji.(c) Subcellular localization of MdRGA2L.Upper panel, green fluorescent protein (GFP); lower panel, MdRGA2L-GFP.Scale bars 16 μm.Data are represented as the mean ± SD.CC, coiled-coil; DAPI, 4,6-diamidino-2phenylindole dihydrochloride; LRR, leucine-rich repeat; NB-ARC, nucleotide-binding adaptor shared by Apaf1, certain R genes and CED4. of SA were modified by the alteration of MdRGA2L expression.The contents of SA and levels of MdICS1 transcript showed a consistent trend with the level of MdRGA2L transcript.In addition, the levels of MdPR1 transcript dramatically increased in the MdRGA2L-OE plants and significantly decreased in the MdRGA2L-Ri plants (Figure 6c,d).

F
MhYTP2 decreased the transcript level of MdRGA2L after inoculation with Colletotrichum fructicola.(a) The transcript levels of MdRGA2L in the wild-type (WT) and 35S::MhYTP2 overexpression lines (OE-1, OE-2, and OE-3).Data are represented as the mean ± SD.Different letters indicate significant differences among the WT and transgenic plants on the same day of different treatments.(b) The R genes whose translation efficiency (TE) are affected by MhYTP2.(c) MdRGA2L protein expression in the 35S::MhYTP2 lines and the WT plants at the start and 6 days after inoculation with C. fructicola.Ponceau S staining shows the loading control.*p < 0.05, **p < 0.01.

F
MdRGA2L enhanced the resistance of apple to Glomerella leaf spot (GLS) by activating the salicylic acid (SA) signalling pathway.(a) Disease severity was recorded 3 days after inoculation with Colletotrichum fructicola in the MdRGA2L-OE, MdRGA2L-Ri, empty vector (EV) control, MdRGA2L-OE + MdDMR6-OE, and MdRGA2L-Ri + SA plants.Scale bars 1 cm.(b) Recorded disease severity and summary statistics of GLS susceptibility 3 days after inoculation with C. fructicola on Fuji MdRGA2L-Ri and vector pK7-expressing leaves.Scale bars 1 cm.(c) The contents of SA in the MdRGA2L-OE, MdRGA2L-Ri, EV control, MdRGA2L-OE + MdDMR6-OE, and MdRGA2L-Ri + SA plants.FW, fresh weight.(d) The transcript levels of MdICS1 and MdPR1 in the MdRGA2L-OE, MdRGA2L-Ri, and EV control leaves.The EV controls are pCambia2300 (2300) and pK7WIWG2D (pK7).Data are represented as the mean ± SD.Different letters indicate significant differences among the wild-type (WT) and transgenic plants on the same day of different treatments.OE, overexpression; Ri, RNA interference.

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I G U R E 7 MdRGA1 (MD07G1015300) has no function in Glomerella leaf spot (GLS) resistance.The changes in the transcript levels of genes encoding CC-NBS-LRR proteins (a), NBS-LRR proteins (b), and TIR-NBS-LRR proteins (c) during the progression of inoculation with Colletotrichum fructicola in the GLS-resistant cultivar Fuji.The ratio values on the left of the heatmap represent the number of transcripts of R genes in response to inoculation with C. fructicola in the GLS-resistant cultivar Fuji at 5 days postinoculation compared with that of the control at day 5.(d) Disease severity recorded 3 days following the inoculation with C. fructicola of the MdRGA1-OE, MdRGA1-Ri, and empty vector (EV) plants.The EV controls are pCambia2300 (2300) and pK7WIWG2D (pK7).Scale bars 1 cm.CC-NBS-LRR, coiled-coil nucleotidebinding site leucine-rich repeat; NBS-LRR, nucleotide-binding site leucine-rich repeat; OE, overexpression; Ri, RNA interference; TIR-NBS-LRR, toll/interleukin-1 receptor.