Comparative transient expression analyses on two conserved effectors of Colletotrichum orbiculare reveal their distinct cell death-inducing activities between Nicotiana benthamiana and melon.

Colletotrichum orbiculare infects cucurbits, such as cucumber and melon (Cucumis melo), as well as the model Solanaceae plant Nicotiana benthamiana, by secreting an arsenal of effectors that suppress the immunity of these distinct plants. Two conserved effectors of C. orbiculare, called NLP1 and NIS1, induce cell death responses in N. benthamiana, but it is unclear whether they exhibit the same activity in Cucurbitaceae plants. In this study, we established a new Agrobacterium-mediated transient expression system to investigate the cell death-inducing activity of NLP1 and NIS1 in melon. NLP1 strongly induced cell death in melon but, in contrast to the effects seen in N. benthamiana, mutations either in the heptapeptide motif or in the putative glycosylinositol phosphorylceramide-binding site did not cancel its cell death-inducing activity in melon. Furthermore, NLP1 lacking the signal peptide caused cell death in melon but not in N. benthamiana. Study of the transient expression of NIS1 also revealed that, unlike in N. benthamiana, NIS1 did not induce cell death in melon. In contrast, NIS1 suppressed flg22-induced reactive oxygen species generation in melon, as seen in N. benthamiana. These findings indicate distinct cell death-inducing activities of NLP1 and NIS1 in these two plant species that C. orbiculare infects.

Anthracnose fungi belonging to the genus Colletotrichum cause severe diseases in a wide range of crop and ornamental plants. Most Colletotrichum species take a hemibiotrophic infection strategy, that is, the pathogen initially grows biotrophically in living host cells and then switches to a destructive necrotrophic phase of infection (Perfect et al., 1999). In the biotrophic stage, it is thought that Colletotrichum fungi synthesize and secrete an arsenal of effectors that suppress host immunity and sustain the viability of host tissues. In the subsequent necrotrophic stage, it is assumed that the pathogen secretes toxins, lytic enzymes, and other types of effectors to kill host cells and gains nutrients to support fungal growth in planta (Gan et al., 2013; Kleemann et al., 2012; O’Connell et al., 2012). Colletotrichum orbiculare causes anthracnose disease in cucurbits such as cucumber and melon, but interestingly, the pathogen can also infect Nicotiana benthamiana, which is distantly related to cucurbits (Shen et al., 2001; Takano et al., 2006).

Some effectors are widely distributed across species (Bart et al., 2012; Irieda et al., 2019; Liu et al., 2019). One typical example is necrosis‐ and ethylene‐inducing‐like proteins (NLPs), which are conserved in many pathogenic bacteria, fungi, and oomycetes (Gijzen & Nürnberger, 2006). Many NLPs are cytotoxic proteins with a strong ability to induce necrosis in eudicot plants such as N. benthamiana via binding to eudicot plant‐specific sphingolipids called glycosylinositol phosphorylceramides (GIPCs) (Lenarčič et al., 2017; Pemberton & Salmond, 2004). The conserved NLP domain, which has the heptapeptide motif GHRHDWE present in its central region, is required for the cell death‐inducing activity (Ottmann et al., 2009; Seidl & Van den Ackerveken, 2019). We recently reported that C. orbiculare expresses a typical NLP protein, named NLP1, in the late infection phase and that the Agrobacterium‐mediated transient expression of NLP1 induces necrosis in N. benthamiana (Azmi et al., 2018). However, it is unclear whether NLP1 can induce cell death in host Cucurbitaceae plants that C. orbiculare naturally infects.

In this study, we investigated the cell death‐inducing activity of NLP1 and various NLP1 mutants in melon (Cucumis melo). For this purpose, we established a new Agrobacterium‐mediated transient expression system in melon using a pEAQ‐HT vector designed to allow quick and efficient production of recombinant proteins in plants (Sainsbury et al., 2009). We transformed pEAQ‐HT (empty vector [EV]) and pEAQ‐HT‐GFP into Agrobacterium tumefaciens GV3101 (pMP90) by electroporation (Koncz & Schell, 1986). The suspension of transformed Agrobacterium was infiltrated (hereafter called agroinfiltration) into melon cotyledons from the abaxial surface using a needleless syringe. We found that MES‐KOH, included in the infiltration buffer, was toxic to melon cotyledons and produced insufficient green fluorescent protein (GFP) production, whereas removal of MES‐KOH from the infiltration buffer significantly improved GFP expression in the melon cotyledon (Figure S1). The GFP production increased further when we changed to Agrobacterium strain GV2260 transformed with pBBRgabT (hereafter called GV2260 [gabT]; Nonaka et al., 2017) instead of GV3101 (pMP90) (Figure S2). Therefore, we decided to use Agrobacterium GV2260 (gabT) suspended in the infiltration buffer without MES‐KOH for the agroinfiltration assay.

We next infiltrated Agrobacterium GV2260 (gabT) with pEAQ‐HT‐NLP1:HA (primers used for plasmid construction are listed in Table S1) into melon cotyledons and N. benthamiana leaves. The infiltrated areas were observed at 5 days postinfiltration. For melon cotyledons, we infiltrated the suspension into the whole area of cotyledons. The transient expression of NLP1 induced necrotic symptoms in both N. benthamiana leaves (Figure 1b,c) and melon cotyledons (Figure 2a,b). In melon cotyledons, on average, 15.6% of the area of cotyledons infiltrated with Agrobacterium carrying pEAQ‐HT‐NLP1:HA displayed yellowish or necrotic symptoms, whereas cotyledons infiltrated with Agrobacterium carrying pEAQ‐HT (EV) did not exhibit such symptoms. These results show that NLP1 exhibits cell death‐inducing activity toward two unrelated plant species (N. benthamiana and melon) that C. orbiculare is able to infect.

FIGURE 1

Cell death‐inducing activity of wild‐type and mutated NLP1 proteins expressed in Nicotiana benthamiana. (a) The amino acid sequences of Colletotrichum orbiculare NLP1 and NLPPya. NLP1 (GenBank: TDZ25257.1) was aligned with the amino acid sequence of NLPPya (GenBank: 3GNU_P) using ClustalW (Thompson et al., 1994). Amino acids of NLPPya in the red box are located within the heptapeptide motif (underlined) and thought to be involved in coordination of the Mg2+ ion. We performed mutational analyses of the corresponding amino acids of NLP1 (D119, H127, and E132). Amino acids in the blue box of NLPPya are involved in binding glycosylinositol phosphorylceramides (GIPCs) in the eudicot plant cell membrane. We also performed mutational analyses of the corresponding amino acids of NLP1 (F181 and N184). The amino acid sequence in the green box is the signal peptide (SP) sequence of NLP1 predicted by SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP/). We used the amino acid sequence of NLPPya lacking the N‐terminal region for the alignment because the crystal structure of the corresponding region and subsequent mutational analyses have been reported (Ottmann et al., 2009). (b) Transient expression assay of the wild‐type and mutated NLP1 proteins in N. benthamiana. N. benthamiana was challenged with Agrobacterium tumefaciens GV2260 (gabT) harbouring pEAQ‐HT (empty vector [EV]), pEAQ‐HT‐NLP1:HA, pEAQ‐HT‐NLP1H127A:HA, pEAQ‐HT‐NLP1D119A/H127A/E132A:HA, pEAQ‐HT‐NLP1F181A:HA, or pEAQ‐HT‐NLP1F181A/N184K:HA. The photograph was taken at 5 days postinfiltration. Similar results were obtained in two additional experiments. (c) Quantitative analysis of the lesion area per infiltration area. Means and standard errors were calculated from three independent experiments. Amino acid abbreviations: A, alanine; D, aspartic acid; E, glutamic acid; F, phenylalanine; H, histidine; K, lysine; N, asparagine

FIGURE 2

Cell death‐inducing activity of wild‐type and mutated NLP1 proteins expressed in melon. (a) The whole area of melon cotyledons was infiltrated with the Agrobacterium tumefaciens strains shown in Figure 1b. The photograph was taken at 5 days postinfiltration (dpi). (b) Quantitative analysis of necrotic lesion development in infiltrated melon cotyledons from (a). ImageJ was used for measuring the ratios of lesion area in infiltrated cotyledons by adjusting the colour threshold. Means and standard errors were calculated from three independent experiments. (c) Protein extracts at 5 dpi from melon cotyledons expressing the wild‐type or mutated NLP1 proteins were analysed by immunoblotting (IB) using anti‐HA (3F10; Roche) as the primary antibody and horseradish peroxidase‐linked anti‐rat IgG (#7077; Cell Signaling Technology) as the secondary antibody

Given the tertiary structure of the NLP of Pythium aphanidermatum (NLPPya), three amino acid residues, H101, D104, and E106, from the central heptapeptide motif GHRHDWE and its associated conserved residue D93 are thought to be involved in the coordination of a Mg2+ ion within the negatively charged cavity exposed at the protein surface (Figure 1a; Ottmann et al., 2009). Supporting this idea, single mutations of D93, H101, and E106 to alanine (D93A, H101A, and E106A, respectively), in two NLPPya homologs, NLPPp from Phytophthora parasitica and NLPPcc from Pectobacterium carotovorum, resulted in the loss of ability to trigger necrosis in N. benthamiana leaves. These results confirmed that these amino acid residues are required for the biological activity of NLP tested in N. benthamiana (Ottmann et al., 2009). Consistently, H127A in the C. orbiculare NLP1, which corresponds to H101A in NLPPya, diminishes cell death‐inducing activity in N. benthamiana (Azmi et al., 2018; Ottmann et al., 2009). To determine whether this mutation also impairs the activity of NLP1 in melon, we constructed pEAQ‐HT‐NLP1H127A:HA to express NLP1:HA carrying H127A (hereafter called NLP1H127A) in melon (Figure 1a). Using this new system, we confirmed that, unlike the wild‐type (WT) NLP1, the expression of NLP1H127A did not result in the development of necrotic lesions in N. benthamiana (Figure 1b,c). Surprisingly, in melon, the expression of NLP1H127A resulted in yellowish and necrotic symptoms, similar to those observed in the WT NLP1 (Figure 2a,b). Western blotting using an anti‐HA antibody showed that NLP1 and NLP1H127A proteins accumulated in melon cotyledons at similar levels (Figure 2c). These findings suggest that H127 of C. orbiculare NLP1 is essential for the cell death‐inducing activity in N. benthamiana, but not in melon.

To extend this finding, we also investigated the effect of triple mutation D119A/H127A/E132A (corresponding to D93A/H101A/E106A in NLPPya) on the cell death‐inducing activity of NLP1 in both melon and N. benthamiana (Figure 1a). For this purpose, we constructed pEAQ‐HT‐NLP1D119A/H127A/E132A:HA (Table S1) and introduced it into the Agrobacterium strain. An agroinfiltration assay revealed that NLP1D119A/H127A/E132A caused yellowish and necrotic symptoms in melon, as seen in WT NLP1 (Figure 2a,b). This finding suggests that these amino acids, located in the central heptapeptide motif GHRHDWE or its neighbour region, are dispensable for the cell death‐inducing activity of NLP1 in melon. Interestingly, in N. benthamiana, NLP1D119A/H127A/E132A induced necrotic lesions to some extent, whereas NLP1H127A did not induce any necrotic lesions (Figure 1b,c).

A recent report has shown that the surface‐exposed GIPCs in eudicot plants are NLPPya toxin receptors that bind to NLPPya H101 and D158, causing a conformational change to facilitate the insertion of NLPPya into the plasma membrane of N. benthamiana (Lenarčič et al., 2017). NLPPya W155A failed to bind to GIPC and exhibited no cytotoxic activity toward N. benthamiana, suggesting that the hydrophobic residue W155 near D158, the GIPC‐binding site, is also involved in the interaction with plant cell membrane. It has also been shown that both NLPPya W155A and NLPPya D158K severely reduce the cytotoxic activity toward two Brassicaceae plants, Arabidopsis thaliana and Brassica oleracea (Lenarčič et al., 2017). We next asked whether the GIPC‐binding ability of NLP1 is related to its cytotoxic activity in melon and N. benthamiana. The amino acid alignment analysis of NLP1 and NLPPya suggested that the corresponding residue of W155 in NLPPya is F181 in NLP1 (Figure 1a), which is consistent with the fact that both amino acids are aromatic and hydrophobic amino acids.

We constructed pEAQ‐HT‐NLP1F181A:HA (Table S1) and introduced it into the Agrobacterium strain. We also constructed pEAQ‐HT‐NLP1F181A/N184K:HA (N184 in NLP1 corresponds to D158 in NLPPya) (Figure 1a) and then generated another Agrobacterium strain harbouring this plasmid. The agroinfiltration assay revealed that NLP1F181A lost cell death‐inducing activity and that NLP1F181A/N184K markedly reduced the activity in N. benthamiana (Figure 1b,c). These observations are consistent with the results of a previous report (Lenarčič et al., 2017). Surprisingly, both NLP1F181A and NLP1F181A/N184K caused yellowish and necrotic symptoms in melon, which contrasted with the case of N. benthamiana (Figure 2b,c). These results suggest that the amino acids tested are involved in binding GIPCs and are required for the cell death‐inducing activity in N. benthamiana, but not in melon. Interestingly, we found that NLP1F181A, but not NLP1F181A/N184K, exhibited greater cytotoxic activity than NLP1 in melon (Figure 2b,c).

Based on the finding that all the tested NLP1 mutants caused cell death in melon, we further investigated the cell death‐inducing activity of NLP1 lacking its signal peptide (hereafter called NLP1∆SP) in N. benthamiana and melon. We found that the transient expression of NLP1∆SP failed to cause cell death in N. benthamiana (Figure 3a), which is consistent with the previous finding that Phytophthora sojae NLP lacking the signal peptide failed to cause cell death in A. thaliana (Qutob et al., 2006). Surprisingly, NLP1∆SP slightly reduced the cell death‐inducing activity in melon but still caused cell death clearly (Figure 3b,c), suggesting that the machineries for NLP1‐triggered cell death are probably distinct between melon and N. benthamiana.

FIGURE 3

NLP1 lacking the signal peptide‐induced cell death in melon but not in Nicotiana benthamiana. (a) Transient expression of NLP1 without its signal peptide (NLP1∆SP) failed to cause cell death in N. benthamiana. N. benthamiana leaves were challenged with Agrobacterium tumefaciens GV2260 (gabT) harbouring pEAQ‐HT‐NLP1:HA and pEAQ‐HT‐NLP1ΔSP:HA to express NLP1 or NLP1ΔSP. The photograph was taken at 5 days postinfiltration (dpi). (b) Transient expression assay of NLP1 and NLP1ΔSP in melon. The whole area of melon cotyledons was infiltrated with each A. tumefaciens strain. The photograph was taken at 5 dpi. (c) Quantitative analysis of necrotic lesion development in infiltrated melon cotyledons from (b)

Next, to gain further insights into whether the cell death‐inducing activity of C. orbiculare effectors differs between N. benthamiana and melon, we focused on another conserved effector, necrosis‐inducing secreted protein 1 (NIS1), of C. orbiculare (Irieda et al., 2019; Yoshino et al., 2012). We previously reported that transient expression of NIS1 caused necrotic lesion formation in N. benthamiana (Yoshino et al., 2012). NIS1 is conserved in a broad range of fungi in both Ascomycota and Basidiomycota, where it targets key kinases such as BAK1/SERK3 and BIK1 that are required for plant pattern‐triggered immunity (PTI) signalling and suppresses multiple PTI responses, including flg22‐triggered generation of reactive oxygen species (ROS) (Irieda et al., 2019). However, the PTI‐suppressing activity of NIS1 in cucurbits remains to be elucidated.

To investigate this further, we constructed pEAQ‐HT‐NIS1:HA (Table S1). We first transiently expressed NIS1 in N. benthamiana and found that the expression of NIS1 caused necrotic lesions in N. benthamiana (Figure 4a,b), a finding that is consistent with our previous report (Yoshino et al., 2012). We then transiently expressed NIS1 in melon cotyledons via agroinfiltration. NIS1 failed to cause cell death in melon cotyledons (Figure 4c). Western blot analysis confirmed the NIS1:HA accumulation in melon cotyledons (Figure 4d). These findings suggest that, in contrast to the observations in N. benthamiana, NIS1 has no cell death‐inducing activity in melon. We next investigated whether the transient expression of NIS1 suppresses flg22‐triggered ROS generation in melon cotyledons. The transient expression of NIS1 markedly reduced flg22‐triggered ROS generation (Figure 4e), suggesting that NIS1 can suppress PTI signalling in melon. A mutated NIS1 that lacks the 30 amino acids at the C‐terminus (hereafter called NIS1∆C30) fails to induce cell death in N. benthamiana but it suppresses flg22‐triggered ROS generation and also associates with BAK1 and BIK1 (Irieda et al., 2019). We investigated the effect of NIS1∆C30 on flg22‐triggered ROS generation in melon and found that NIS1∆C30 reduced flg22‐triggered ROS generation (Figure S3), suggesting that NIS1 suppresses flg22‐triggered ROS generation in both melon and N. benthamiana in a similar way.

FIGURE 4

NIS1 did not cause cell death but suppressed reactive oxygen species (ROS) generation in melon. (a) Nicotiana benthamiana leaves were challenged with Agrobacterium tumefaciens GV2260 (gabT) harbouring pEAQ‐HT (empty vector [EV]) or pEAQ‐HT‐NIS1:HA (NIS1). The photograph was taken at 5 days postinfiltration (dpi). (b) Quantitative analysis of the lesion area per infiltration area in N. benthamiana. Means and standard errors were calculated from three independent experiments. (c) NIS1 did not cause development of necrotic lesions in melon. The photograph was taken at 5 dpi. (d) Protein extracts at 5 dpi from melon cotyledons expressing NIS1:HA were analysed by immunoblotting (IB) using an anti‐HA antibody. (e) Assay of flg22‐triggered ROS generation in melon. Melon cotyledons were infiltrated with A. tumefaciens GV2260 (gabT) harbouring pEAQ‐HT (empty vector [EV]) or pEAQ‐HT‐NIS1:HA. At 5 dpi, leaf discs were taken from cotyledons and incubated overnight in distilled water (DW) in the dark, 1 µM flg22 or DW was added, and ROS generation was measured (Irieda et al., 2019). Data are given as relative light units (RLU) and represent the mean ± SE (n = 12). Similar results were obtained in another independent experiment

In this study, we established a new efficient system of transient protein expression in melon. Using this new system, we found that two widely conserved fungal effectors, NLP1 and NIS1, from C. orbiculare exhibit distinct activities in melon and N. benthamiana, both of which this pathogen infects.

In the NLP1 study, we performed mutational analysis on the putative GIPC‐binding sites of NLP1 in N. benthamiana. Consistent with a previous report (Lenarčič et al., 2017), our results suggest that NLP1 F181 is involved in binding to the cell membrane‐exposed GIPCs in N. benthamiana (Figure 1b,c). However, the F181A mutation did not cancel the cell death‐inducing activity of NLP1 in melon; conversely, the cell death‐inducing activity of NLP1F181A was increased in melon (Figure 2a,b).

Cytotoxic NLPs, including NLP1, contain a heptapeptide motif (GHRHDWE) that has been shown to be critical for the cytotoxic activity of NLPs in N. benthamiana (Ottmann et al., 2009). The results of our study using NLP1H127A also support this finding. Interestingly, NLP1D119A/H127A/E132A recovered the cytotoxic activity in N. benthamiana to some extent (Figure 1b,c). Although it remains to be elucidated how NLP1D119A/H127A/E132A recovered this activity, this finding suggests that D119A and E132A changed the tertiary structure of NLP1H127A, which resulted in the partial recovery of the structure required for the cytotoxic activity in N. benthamiana.

Importantly, we found that the point mutations in NLP1 described above did not cancel cell death induction in melon, in contrast to N. benthamiana. Why did the tested mutations not abolish the cell death‐inducing activity of NLP1 in melon? Surprisingly, we found that NLP1∆SP induced cell death in melon, whereas NLP1∆SP completely lacked the cell death‐inducing activity in N. benthamiana (Figure 3). This finding suggests that apoplastic localization of NLP1 is critical for the cell death induction in N. benthamiana, whereas cytoplasmic NLP1 is able to trigger cell death in melon, that is, the machineries for NLP1‐triggered cell death are fundamentally distinct between N. benthamiana and melon. This idea is further supported by the finding that the point mutations in NLP1 tested in this study did not cancel cell death induction in melon, in contrast to N. benthamiana.

We recently reported that C. orbiculare transformants constitutively expressing NLP1 failed to develop lesions in multiple cucurbits, including melon (Azmi et al., 2018). However, in contrast to cucurbits, C. orbiculare constitutively expressing NLP1 caused lesions in N. benthamiana (Azmi et al., 2018), although the transient expression of NLP1 caused severe necrotic lesions in N. benthamiana (Figure 1b,c). These findings suggest the possibility that cytoplasmic NLP1 is recognized by unidentified machineries of melon, leading to cell death responses together with activation of immune responses effective to C. orbiculare. In the inoculation of C. orbiculare transformants constitutively expressing NLP1 on melon, a part of NLP1 secreted by the pathogen might enter into melon cells and strongly activate the host immunity.

In our study, the expression of C. orbiculare NIS1 in melon via the new transient expression system showed that NIS1 did not exhibit the ability to induce cell death in melon, in contrast to N. benthamiana, even though the pathogen can infect both plants. NIS1 and NIS1∆C30 suppressed the flg22‐elicited ROS burst in both melon (this study) and N. benthamiana (Irieda et al., 2019), suggesting that the ability of NIS1 to suppress PTI signalling is conserved in both plants. We previously reported that NIS1‐induced cell death depends on both SGT1 and HSP90 (Yoshino et al., 2012), which suggests that NIS1‐induced cell death may be triggered via the recognition by an unidentified resistance (R) protein. Therefore, melon may not have the R protein for NIS1 recognition.

Overall, our results show that the cell death‐inducing activities and underlying mechanisms of the two C. orbiculare effectors differ between N. benthamiana and melon, both of which can be infected by this pathogen. For a greater understanding of the effector function of plant pathogens, it will be important to establish new transient expression systems in corresponding host plants in addition to the commonly used N. benthamiana.

FIGURE S1 Agroinfiltration buffer without MES‐KOH improved protein expression in melon. Agrobacterium tumefaciens GV3101 (pMP90) harbouring pEAQ‐HT (empty vector [EV]) or pEAQ‐HT‐GFP was grown in Luria Bertani (LB) medium containing kanamycin, rifampicin, and gentamycin (each 50 µg/ml) overnight. The cells were harvested by centrifugation and suspended in infiltration buffer with MES‐KOH (10 mM MES‐KOH pH 5.6, 10 mM MgCl2, and 200 µM acetosyringone) or infiltration buffer without MES‐KOH (10 mM MgCl2 and 200 µM acetosyringone) and adjusted to an OD600 value of 0.3. The A. tumefaciens suspensions were infiltrated into 8‐day‐old melon cotyledons. At 5 days postinfiltration, proteins were extracted from the cotyledons by homogenizing with extraction buffer (50 mM Tris‐HCl pH 7.4, 150 mM NaCl, 5% glycerol, 0.5% Triton X‐100) on ice and centrifuged at 21,000 × g for 10 min at 4 °C to remove cell debris. The Bradford protein assay was conducted, and the protein concentration was calculated. The samples were then subjected to immunoblotting (IB). Anti‐GFP (GFP[B‐2]; Santa Cruz Biotechnology) was used as the primary antibody and horseradish peroxidase‐linked anti‐mouse IgG antibody (#7076; Cell Signaling Technology) was used as the secondary antibody. Ponceau S staining buffer (5% wt/vol in glacial acetic acid) was used to examine the sample loading in the immunoblotting membrane. Similar results were obtained in two additional experiments. MES, 2‐(N‐morpholino)ethanesulfonic acid

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FIGURE S2 Infiltration of Agrobacterium tumefaciens GV2260 (gabT) significantly increased protein expression in melon. A. tumefaciens GV2260 (gabT) was grown in Luria Bertani (LB) medium containing kanamycin, rifampicin, gentamycin, and carbenicillin (each 50 µg/ml), whereas A. tumefaciens GV3101 (pMP90) was grown as described in Figure S1. Using the protocol without MES‐KOH buffer described in Figure S1, the suspension of A. tumefaciens GV3101 (pMP90) harbouring pEAQ‐HT‐GFP or A. tumefaciens GV2260 (gabT) harbouring pEAQ‐HT‐GFP was infiltrated into melon cotyledons. At 5 days postinfiltration, proteins were extracted from the infiltrated cotyledons, and the extracted samples were subjected to immunoblotting (IB). For each Agrobacterium line, two independent samples were loaded. The GFP expression level was generally higher in GV2260 (gabT) than in GV3101 (pMP90). Similar results were obtained in two additional experiments

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FIGURE S3 NIS1 lacking the C‐terminal 30 amino acids suppressed flg22‐triggered ROS generation in melon. (a) Nicotiana benthamiana leaves were challenged with Agrobacterium tumefaciens GV2260 (gabT) harbouring pEAQ‐HT (empty vector [EV]), pEAQ‐HT‐NIS1:HA (NIS1), or pEAQ‐HT‐NIS1ΔC30:HA (NIS1ΔC30). The photograph was taken at 5 days postinfiltration (dpi). (b) Assay of flg22‐triggered ROS generation in melon. Melon cotyledons were infiltrated with A. tumefaciens GV2260 (gabT) harbouring pEAQ‐HT (EV), pEAQ‐HT‐NIS1:HA (NIS1), or pEAQ‐HT‐NIS1ΔC30:HA (NIS1ΔC30). At 5 dpi, leaf discs (diameter, 6 mm) were taken from infiltrated melon cotyledons (note that it is important to take leaf discs at positions close to the agroinfiltration sites to ensure effective expression of target protein). Leaf discs were then incubated in the distilled water for 17 hr under dark conditions. Next, 1 μM flg22 was added, and ROS generation was measured (Irieda et al., 2019). Data are given as relative light units (RLU) and represent the mean ± SE (n = 12). Similar results were obtained in two additional experiments

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TABLE S1 Primers used for plasmid construction in this study

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