The clade III subfamily of OsSWEETs directly suppresses rice immunity by interacting with OsHMGB1 and OsHsp20L

Summary

The clade III subfamily of OsSWEETs includes transmembrane proteins necessary for susceptibility to bacterial blight (BB). These genes are targeted by the specific transcription activator‐like effector (TALE) of Xanthomonas oryzae pv. oryzae and mediate sucrose efflux for bacterial proliferation. However, the mechanism through which OsSWEETs regulate rice immunity has not been fully elucidated. Here, we demonstrated that the cytosolic carboxyl terminus of OsSWEET11a/Xa13 is required for complementing susceptibility to PXO99 in IRBB13 (xa13/xa13). Interestingly, the C‐terminus of ZmXa13, the maize homologue of OsSWEET11a/Xa13, could perfectly substitute for the C‐terminus of OsSWEET11a/Xa13. Furthermore, OsSWEET11a/Xa13 interacted with the high‐mobility group B1 (OsHMGB1) protein and the small heat shock‐like protein OsHsp20L through the same regions in the C‐terminus. Consistent with the physical interactions, knockdown or knockout of either OsHMGB1 or OsHsp20L caused an enhanced PXO99‐resistant phenotype similar to that of OsSWEET11a/OsXa13. Surprisingly, the plants in which OsHMGB1 or OsHsp20L was repressed developed increased resistance to PXO86, PXO61 and YN24, which carry TALEs targeting OsSWEET14/Xa41 or OsSWEET11a/Xa13. Additionally, OsHsp20L can interact with all six members of clade III OsSWEETs, whereas OsHMGB1 can interact with five other members in addition to OsSWEET12. Overall, we revealed that OsHMGB1 and OsHsp20L mediate conserved BB susceptibility by interacting with clade III OsSWEETs, which are candidates for breeding broad‐spectrum disease‐resistant rice.

Introduction

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating rice diseases and can result in up to 50% yield losses under favourable field conditions (Niño‐Liu et al., 2006). Breeding BB‐resistant varieties is regarded as one of three ‘E’ strategies, including effective, economical and eco‐friendly strategies (Jiang et al., 2020). More than 40 resistance (R) genes conferring genetic dominance or recessive have been identified from wild species or cultivar varieties. Among them, 18 genes have been cloned and classified into five categories based on their encoded proteins (Jiang et al., 2020). Three categories are encoded by dominant R genes, including the receptor‐like kinase (RLK) category, which is composed of Xa21 (Song et al., 1995), Xa3/Xa26 (Sun et al., 2004) and Xa4 (Hu et al., 2017); the nucleotide‐binding leucine‐rich repeat (NB‐LRR) receptor category, which consists of Xa1, Xa1‐2, Xa2, Xa14, CGS‐Xo1 (Ji et al., 2020; Zhang et al., 2020); and the executor category, which consists of Xa7 (Chen et al., 2021; Luo et al., 2021), Xa10 (Tian et al., 2014), Xa23 (Wang et al., 2015) and Xa27 (Gu et al., 2005). The other two categories are encoded by recessive R genes, the dominant alleles of which encode the susceptible proteins xa5, which encodes the mutated gamma subunit of transcription factor IIA (Iyer and McCouch, 2014; Jiang et al., 2016a); xa13 (Chu et al., 2006; Yang et al., 2006); xa25 (Liu et al., 2011); and xa41 (Hutin et al., 2015), which harbour mutations in the promoters to escape pathogen‐inducible expression manipulated by specific transcription activator‐like effectors (TALEs) secreted from Xoo (Boch et al., 2009; Xu et al., 2022) and encode sugars that will eventually be exported as transporter (SWEET) proteins (Chen et al., 2010).

SWEETs are seven transmembrane sugar transporters involved in the physiological development of multifarious plants (Chen et al., 2010). There are 22 SWEET proteins belonging to four subfamilies in rice (Wu et al., 2022a). The clade III subfamily, consisting of six members (OsSWEET11a, OsSWEET11b, OsSWEET12‐OsSWEET15), is known as a BB‐susceptible gene activated by each cognate TALE (Breia et al., 2021; Streubel et al., 2013; Wu et al., 2022a). Several models of TALE‐OsSWEET, including PthXo1/Tal6b‐OsSWEET11a/Xa13, PthXo2‐OsSWEET13/Xa25 and PthXo3/AvrXa7/Tal5‐OsSWEET14/Xa41, are well established for rice‐Xoo interactions (Antony et al., 2010; Xu et al., 2023; Yang et al., 2006; Zhou et al., 2015). Additionally, activation of OsSWEET12 and OsSWEET15 with designed artificial TALEs (aTALEs) in PXO99a could restore the susceptibility of rice cultivar IRBB13 to recessive xa13 (Streubel et al., 2013). The substitution of each TALE or aTALEs implies a conserved susceptibility role for all clade III OsSWEET members. Additionally, gene editing of the TALE binding elements (EBEs) of OsSWEET11a/Xa13 and OsSWEET14/Xa41 resulted in broad‐spectrum resistance in Xoo strains (Oliva et al., 2019; Xu et al., 2019). In addition to BB susceptibility, OsSWEET11a/Xa13 and OsSWEET14/Xa41 negatively and positively regulate rice resistance to sheath blight, respectively (Gao et al., 2018; Kim et al., 2020). Coincidentally, clade III SWEETs transport sugars, hexanose and GA and cooperatively/redundantly function in seed filling (Kanno et al., 2016; Wu et al., 2022a; Yang et al., 2018). However, only OsSWEET11a/Xa13 has been shown to physically interact with the copper transporter COPT1, COPT5 and the thylakoid membrane‐bound ascorbate peroxidase OsAPX8 to reduce the distribution of copper ions in xylem vessels and repress H₂O₂‐scavenging reactions to increase susceptibility in rice (Jiang et al., 2016b; Yuan et al., 2010). To date, the biochemical evidence for exploring the susceptibility mechanism of clade III OsSWEETs remains unclear.

High‐mobility group box (HMGB) proteins have a distinctive DNA‐binding motif termed the HMG‐box domain, which confers non‐sequence‐specific interactions with linear DNA and structure‐specific binding to distorted DNA sites (Stemmer et al., 2003). These proteins act as architectural factors facilitating the assembly of nucleoprotein complexes, which, for instance, regulate transcription and recombination (Bustin, 1999; Thomas and Travers, 2001). In mammals, the HMGB1 protein not only binds DNA but also acts as a damage‐associated molecular pattern (DAMP) when it is released from necrotic cells into the extracellular space to trigger an immune response (Andersson and Tracey, 2011; Lotze and Tracey, 2005; Yang et al., 2015). Plants also contain HMGB proteins that bind to various DNA structures. For example, all four maize HMGB proteins have been shown to bind DNA minicircles and superhelical DNA (Ritt et al., 1998). Maize HMGB1 and HMGB5 can interact with and stimulate the DNA‐binding effect of the transcription factor Dof2 (Grasser et al., 2007); there are six HMGB proteins in rice. OsHMGB1 can bind four‐way junction DNA and DNA minicircles (Wu et al., 2003) and positively regulate phosphate homeostasis by binding to promoters and activating the expression of a series of phosphate starvation‐responsive (PSR) genes (Wang et al., 2023). However, the involvement of the HMGB protein in the immune response has rarely been reported in plants. In Arabidopsis, AtHMGB3 is regarded as a DAMP that is released into the apoplast after infection by B. cinerea and triggers hallmark immune responses via AtBAK1 and AtBKK1 (Choi et al., 2016). In contrast, the function of the HMGB protein in the rice immune response is unknown.

Heat shock proteins (HSPs), which function as molecular chaperones, are ubiquitous proteins found in plant and animal cells (Waters, 2013). Based on their approximate molecular weights, HSPs are grouped as HSP100s, HSP90s, HSP70s, HSP60s and HSP20s or small HSPs (sHSPs, approximately 12–42 kDa). Recently, sHSPs have been emphasized for their ability to improve immunity by inducing pathogenesis‐related (PR) gene expression and inhibiting the growth and infective ability of bacteria, fungi or viruses (Wu et al., 2022b). Overexpression of OsHsp18.0‐CI enhanced the resistance of transgenic rice to bacterial leaf streak and BB by priming an enhanced basal defence (Ju et al., 2017). Overexpression of VvHSP24 in Arabidopsis thaliana increased resistance to B. cinerea via constitutive expression of the SA‐responsive PR1, PR2 and PR5 genes (Li et al., 2021). The expression of the soybean gene GmHsp22.4 was strongly induced in the resistant soybean genotype but repressed in the susceptible genotype after inoculation with the nematode Meloidogyne javanica. Moreover, the multiplication and infective potential of the nematode were significantly reduced by heterologous overexpression in Arabidopsis (Hishinuma‐Silva et al., 2020).

In addition to the conserved transmembrane domains, OsSWEETs also contain a short cytosolic domain at the C‐terminus, which has not been functionally characterized. To reveal the functions of these genes, we selected OsSWEET11a/Xa13 as an example. In this study, we genetically determined that the cytosolic domain was required for full susceptibility of OsSWEET11a/Xa13, which could be functionally replaced by the C‐terminus of its homologue. Furthermore, we identified two new interactors of OsSWEET11a/Xa13 through its C‐terminus, OsHMGB1 (LOC_Os06g51220) and OsHsp20L (LOC_Os01g55270). Interestingly, OsHMGB1 and OsHsp20L could interact with most of the other C‐termini of clade III OsSWEETs in addition to OsSWEET11a/Xa13. Accordingly, plants with repressed OsHMGB1 and OsHsp20L show broad‐spectrum resistance to BB. Thus, our study reveals a new conserved BB susceptibility mechanism mediated by clade III OsSWEETs. Two interactors play a negative role in rice BB resistance and are candidates for breeding broad‐spectrum disease‐resistant rice plants.

Results

The carboxyl terminus of OsSWEET11a/Xa13 is essential for susceptibility

Rice contains six members of the clade III SWEET proteins that share a conserved transmembrane region and a variable unstructured region in the carboxyl terminus (Figure S1a–c). OsSWEET11a/Xa13 contain 307 amino acids (Chu et al., 2006; Yuan et al., 2010) that share the highest identity (80.36%) with a maize homologue and are temporarily referred to as ZmSWEET11a (Zm00001d031647) or ZmXa13 (Figure S1a). Further analysis of OsSWEET11a/Xa13 and ZmXa13 revealed 90.45% and 39.58% identity, respectively, between the putative transmembrane region and the C‐terminus predicted in the cytoplasm (Figure 1a). The MtN3.1 domain (51 to 101 amino acids) of OsSWEET11a/Xa13 was shown to be associated with COPT1 and COPT5 to rescue the copper uptake‐deficient yeast mutant MPY17 (Yuan et al., 2010). We found that one amino acid residue mutant in the MtN3.1 domain of ZmXa13 lost its interaction with COPT5 (Figure S2a). Additionally, the amino acid residue mutant in the ZmMtN3.1 domain of ZmXa13 abolished the ability of this gene to transport copper in double mutants of ctr1 ctr3 yeast (Figure S2b).

Figure 1.

The carboxyl terminus of OsSWEET11a/Xa13 is required for susceptibility to bacterial blight. (a) Amino acid sequence alignment of rice OsSWEET11a/Xa13 and the maize homologous ZmXa13 protein. (b) The relative expression level of transgenes induced by PXO99 in C‐terminus truncated of OsSWEET11a/Xa13 transgenic lines (NR‐9 and NR‐13), ZmXa13 gene transgenic lines (ZR‐1 and ZR‐2), lines of chimeric gene containing N‐terminal of OsSWEET11a/Xa13 and C‐terminus of ZmXa13 (ONR‐12 and ONR‐14) and lines of chimeric gene containing N‐terminal of ZmXa13 and C‐terminus of OsSWEET11a/Xa13 (ZNR‐3 and ZNR‐9) under pOsSWEET11a in bacterial blight (BB) resistant variety IRBB13. (c–d) Disease symptoms (c) and lesion length (d) of the above transgenic lines caused by PXO99 at 14 days post inoculation (dpi). The data are presented as the means ± SDs (n = 15). The asterisks above the columns represent the significant differences between wild‐type IRBB13 and susceptible IR24 or transgenic lines (*P ≤ 0.05; **P ≤ 0.01).

To determine the function of the C‐terminus of OsSWEET11a/Xa13 or ZmXa13, a C‐terminal truncated OsSWEET11a/Xa13 of Xa13^1‐220 and the full coding sequence (CDS) of ZmXa13 were inserted into a pCAMBIA1300 vector under the promoter of the dominant gene OsSWEET11a/Xa13 (Figure S2c). Transgenic plants harbouring pOsSWEET11a:Xa13 ^1‐220 (NR) or pOsSWEET11a:ZmXa13 (ZR) in the resistant variety IRBB13 exhibited an increase in lesion length after inoculation with the Xoo strain PXO99 in the T₁ generation (Figure S3a,b). Two independent lines of each transformation (NR‐9, NR‐13 and ZR‐1, ZR‐2) were selected for further analysis in the T₂ generation; these plants exhibited moderate Xa13 ^1‐220 or ZmXa13 expression after inoculation with PXO99 (Figure 1b). Additionally, compared with the resistant control, the transgenic plants that expressed Xa13 ^1‐220 or ZmXa13 had slightly longer lesion lengths (Figure 1c,d). Indeed, the truncated Xa13^1‐220, but not ZmXa13 could rescue copper transport together with COPT1 and COPT5 in the ctr1 ctr3 yeast (Figure S2b). These data suggest that both the copper transporter function of the Xa13^1‐220 region and the C‐terminus of ZmXa13 contribute to susceptibility but cannot fully replace OsSWEET11a/Xa13 during the interaction between rice and PXO99. In addition, the C‐terminus of OsSWEET11a/Xa13 is essential for full susceptibility.

To assess the involvement of the C‐terminal region in rice resistance to PXO99, we altered the C‐terminus of OsSWEET11a/Xa13 and ZmXa13 and subsequently expressed each chimeric protein from pOsSWEET11a:Xa13^1‐220‐ZmXa13^221‐310 (ONR) and pOsSWEET11a:ZmXa13^1‐220‐Xa13^221‐307 (ZNR) in IRBB13 (Figure S2c). The expression of two chimeric protein‐encoding genes was up‐regulated after infection with PXO99 (Figure 1b). Segregation of ONR and ZNR was tested in the T₁ generation (Figure S3c,d). The ONR (ONR‐12 and 14) and ZNR (ZNR‐3 and 9) lines exhibited significantly increased susceptibility, which resulted in longer lesion lengths than did the IRBB13, NR and ZR lines in the T₂ generation (Figure 1c,d). These data suggest that both the transmembrane domain in the amino terminus and the C‐terminus of OsSWEET11a/Xa13 are essential for full susceptibility to infection during the rice‐Xoo interaction.

OsSWEET11a/Xa13 interact with the high‐mobility group box 1 protein

To elucidate the molecular mechanisms of the C‐terminal region of OsSWEET11a/Xa13 during the rice‐Xoo interaction, we used the C‐terminus of OsSWEET11a/Xa13 as bait to screen a Y2H cDNA library from the susceptible rice variety IR24 inoculated with PXO99. Six cDNA clones, including a high‐mobility group Box 1 protein (OsHMGB1) and a small heat shock‐like protein (OsHsp20L), were identified as putative OsSWEET11a/Xa13 interactors. We first verified the interaction between the C‐terminus of OsSWEET11a/Xa13 and full‐length OsHMGB1 by Y2H (Figure 2a), glutathione S‐transferase (GST)‐mediated pull‐down (Figure 2b), co‐IP (Figure 2c) and bimolecular florescence complementation (BiFC) assays in N. benthamiana plants (Figure 2d). According to the YFP fluorescence signals, the interaction mostly occurred on the plasma membrane and in the cytoplasm (Figure 2d). These results demonstrated that OsHMGB1 physically interacts with the C‐terminus of OsSWEET11a/Xa13 both in vitro and in vivo.

Figure 2.

OsSWEET11a/Xa13 interacts with OsHMGB1 in vivo and in vitro. (a) Identification of the C‐terminus of the OsSWEET11a/Xa13‐interacting protein OsHMGB1 via a yeast two‐hybrid (Y2H) assay. (b–d) The interaction between OsSWEET11a/Xa13^221‐307 and OsHMGB1 was validated by GST pull‐down (b), co‐IP (c) and bimolecular florescence complementation (BiFC) (d) assays. OsSWEET11a/Xa13^221‐307 indicate that the C‐terminus ranges from amino acids 221 to 307. Anti‐His and anti‐myc antibodies were used for detection of the tagged proteins via western blotting. Scale bars, 50 μm.

To reveal the motif at the C‐terminus of OsSWEET11a/Xa13 that determines the interaction with OsHMGB1, we generated a series of truncated constructs and performed Y2H with OsHMGB1. As shown in Figure S4, OsHMGB1 interacted with Xa13^230‐307 as well as with Xa13^173‐255 and Xa13^173‐274, suggesting that there may be a motif ranging from amino acid 230 to 255 involved in the interaction with OsHMGB1. Consistently, the C‐terminus of ZmXa13^221‐310 also interacts with OsHMGB1 (Figure S4).

OsHMGB1 acts as a negative regulator of the rice‐Xoo interaction

Previous studies have shown that constitutive expression of OsSWEET11a/Xa13, COPT1 or COPT5 does not significantly affect resistance to PXO99 in IRBB13 (Yuan et al., 2009, 2010), while RNA interference (RNAi) or gene knockout via the CRISPR of susceptible dominant OsSWEET11a/Xa13 confers dramatically enhanced resistance to PXO99 (Chu et al., 2006; Kim et al., 2019). To investigate whether OsHMGB1 participates in OsSWEET11a/Xa13‐mediated BB susceptibility, we performed a series of genetic assays. First, OsHMGB1 transcript levels were measured in both susceptible and resistant rice leaves of IR24 and IRBB13 after inoculation with PXO99. The results showed that OsHMGB1 was initially activated at 6 hpi in both rice varieties but was expressed at markedly greater levels in susceptible IR24 plants than in resistant IRBB13 plants (Figure 3a). Second, transgenic OsHMGB1‐RNAi and OsHMGB1‐OE plants were generated from the susceptible japonica rice variety Zhonghua 11 (ZH11). All the obtained T₀ transgenic plants were inoculated with PXO99 at the booting stage for disease index investigation. Ten of the twelve transgenic plants with suppressed OsHMGB1 expression presented increased resistance to PXO99 compared to that of wild‐type ZH11 and the transgene‐negative individuals (Figure S5a), and the increased resistance was significantly correlated with a reduced OsHMGB1 transcription level (r = 0.830, n = 12; P < 0.01). In contrast, the increased expression of OsHMGB1 in OsHMGB1‐OE plants caused increased susceptibility to PXO99 compared with that in WT plants (Figure S5b), and the reduced resistance was correlated with increased OsHMGB1 transcription (r = 0.554, n = 12, P < 0.05). Three independent OsHMGB1 lines (RNAi‐14, RNAi‐23 and RNAi‐33) that presented significantly reduced OsHMGB1 transcript levels and three OsHMGB1‐OE lines (OE‐15, OE‐16 and OE‐24) were selected for further analysis of the T₁ generation. Compared with those of ZH11, the relative lesion lengths of these two strains cosegregated with the transgenic events identified by PCR (Figure S6a,b). These results were further confirmed in T₂ generation plants. Similarly, compared with those of the WT plants, the average lesion length caused by PXO99 was 2.86 ± 0.51 cm for the two OsHMGB1‐RNAi plants and 8.33 ± 1.08 cm for the two OsHMGB1‐OE plants (Figure 3b–d). Furthermore, the average bacterial population on the rice leaves was significantly lower (P < 0.05) for the OsHMGB1‐RNAi plants and significantly greater (P < 0.05) for the OsHMGB1‐OE plants than for the WT plants at 8–12 dpi (Figure 3e). Third, we generated OsHMGB1‐RNAi and OsHMGB1‐OE plants in the resistant IRBB13 background. After inoculation with PXO99, we found that suppressing the OsHMGB1 transcript did not affect the resistance of IRBB13 to PXO99, but overexpressing OsHMGB1 could compromise xa13‐mediated resistance to PXO99 (Figure 3f–h; Figure S7a,b). Fourth, we also performed knockout of the OsHMGB1 gene in ZH11 using the CRISPR/Cas9 system. Transgenic plants with a single base deletion (hb1‐1) or insertion (hb1‐5) in the second exon of the OsHMGB1 gene were obtained (Figure S8). The lesion length was shorter in both mutant lines than in the WT after inoculation with PXO99 (Figure 4a). Overall, these data suggest that OsHMGB1 acts as a negative regulator of rice resistance to Xoo PXO99.

Figure 3.

OsHMGB1 negatively regulates rice resistance to bacterial blight. (a) Relative expression level of OsHMGB1 after inoculation with PXO99 in the resistant near‐isogenic line IRBB13 and susceptible line IR24. (b–e) Evaluation of OsHMGB1 expression (b), disease symptoms (c), lesion length (d) and bacterial growth curve (e) caused by PXO99 in OsHMGB1‐suppressing lines (RNAi‐14 and RNAi‐23) and overexpression lines (OE‐16 and OE‐24) of the ZH11 background. (f, g) The lesion lengths (f) and the relative expression level of OsHMGB1 (g) in OsHMGB1‐suppressed and ‐overexpressing lines on the IRBB13 background. (h) Bacterial growth curve of PXO99 in OsHMGB1 transgenic lines in the IRBB13 background. The asterisks above the columns represent the significant differences between the wild‐type rice and OsHMGB1 transgenic rice lines (*P ≤ 0.05; **P ≤ 0.01).

Figure 4.

OsHMGB1 suppression plays a role in broad‐spectrum resistance in Xoo strains. (a–c) Statistics of disease symptoms (upper) and lesion lengths (lower) caused by PXO99 (a), PXO86 (b) and YN24 (c) in OsHMGB1 suppression lines (RNAi‐14 and RNAi‐23) and knockout lines generated by CRISPR (hb1‐1 and hb1‐5) in the ZH11 background. (d–f) Lesion lengths affected by PXO61 (d), PXO86 (e) and YN24 (f) in OsHMGB1‐suppressed lines in the IRBB13 background. The asterisks above the columns represent the significant differences between the wild‐type rice and the OsHMGB1 suppression or gene editing lines (*P ≤ 0.05; **P ≤ 0.01).

OsHMGB1 confers broad‐spectrum resistance to Xoo strains

Knockout or knockdown of OsSWEET11a/Xa13 confers race‐specific resistance only to Xoo PXO99 (Chu et al., 2006; Kim et al., 2019). Animal HMGB proteins orchestrate a variety of functions involved in immunity (Lotze and Tracey, 2005). To test whether OsHMGB1 is involved in the response to other Xoo strains, we inoculated two lines, OsHMGB1‐RNAi (RNAi‐14 and RNAi‐23) and two other gene editing lines (hb1‐1 and hb1‐5), with other Xoo race strains, namely, PXO86, PXO61 and YN24, in the ZH11 background. All four lines showed enhanced resistance to PXO86, PXO61 and YN24, with shorter lesion lengths than those of ZH11 (Figure 4b,c; Figure S9). Consistent with the findings in the ZH11 RNAi lines, two RNAi lines of IRBB13 (RNAi‐7 and RNAi‐9) also exhibited enhanced broad‐spectrum resistance to PXO86, PXO61 and YN24 (Figure 4d–f). Additionally, the wild‐type ZH11 carried the OsSWEET13/xa25 which resistant to the Xoo strain carrying PthXo2 (Xu et al., 2019). The increase in resistance to more race strains by OsHMGB1 knockout or knockdown plants indicates that OsHMGB1 may be a general immune factor involved in BB resistance.

Clade III OsSWEETs engineered with the OsSWEET11a/Xa13 promoter overcome the resistance of xa13

Three out of the six clade III OsSWEETs were identified as targets of cognate TALEs from their original strains (Xu et al., 2022). All TALEs or aTALEs for the cognate clade III OsSWEETs could help the PXO99A strain overcome the resistance of xa13 and increase susceptibility to resistant IRBB13 (Streubel et al., 2013). However, the introduction of TALEs or aTALEs is not limited to activating the expression of cognate OsSWEETs. Exploring the redundancy function of OsSWEETs in overcoming or preventing xa13‐mediated resistance is still poorly understood. Here, we expressed the OsSWEET12, OsSWEET13, OsSWEET14 and OsSWEET15 genes under the promoter of OsSWEET11a/Xa13 in the IRBB13 background. Three independent lines for each construct were identified for cosegregation with enhanced susceptibility to PXO99 in the T₁ generation (Figure S10a–d). Furthermore, the average lesion length of each of the two lines caused by PXO99 was measured for P12 (pOsSWEET11a/Xa13:OsSWEET12), P13 (pOsSWEET11a/Xa13:OsSWEET13), P14 (pOsSWEET11a/Xa13:OsSWEET14) and P15 (pOsSWEET11a/Xa13:OsSWEET15) in the T₂ generation. Compared with those of 2.50 ± 0.58 cm for the resistant IRBB13 (WT) line and 17.38 ± 3.06 cm for the IR24 line, 9.35 ± 2.27 cm and 8.79 ± 1.88 cm for the two P12 lines; 10.06 ± 3.70 cm and 9.36 ± 2.04 cm for the two P13 lines; 11.85 ± 2.21 cm and 12.97 ± 1.79 cm for the two P14 lines; and 9.19 ± 1.75 cm and 9.90 ± 2.14 cm for the two P15 lines, which showed dramatically enhanced susceptibility to PXO99 (Figure 5). These results suggest that the expression of all the clade III SWEET genes induced by PthXo1 can overcome the resistance of xa13 to PXO99 and that these genes play a conserved role in regulating BB resistance.

Figure 5.

Clade III OsSWEETs derived from the pSWEET11a/Xa13 promoter can restore rice susceptibility to PXO99 via IRBB13. (a, b) Disease symptoms (a) and lesion lengths (b) caused by PXO99 on transgenic rice plants harbouring OsSWEET12 (P12‐3 and P12‐9), OsSWEET13/Xa25 (P13‐4 and P13‐5), OsSWEET14/Xa41 (P14‐1 and P14‐2) and OsSWEET15 (P15‐3 and P15‐5) under the control of the OsSWEET11a/Xa13 promoter in the IRBB13 background. The asterisks above the columns represent the significant differences between the wild‐type IRBB13 and clade III OsSWEETs transgenic lines (**P ≤ 0.01).

OsHMGB1 interacts with five clade III OsSWEET proteins

Knockout or knockdown of the OsHMGB1 gene confers resistance to more Xoo strains than does the xa13 gene, and the clade III OsSWEETs, which can replace each other, have redundant functions, suggesting possible interactions between OsHMGB1 and clade III SWEET proteins. Indeed, in a Y2H assay, we found that OsHMGB1 interacted with the C‐terminus of OsSWEET11b, OsSWEET13, OsSWEET14 and OsSWEET15 but not with OsSWEET12 or a Clade IV member of OsSWEET16 (Figure 6a; Figure S11a). Next, the direct interactions between OsHMGB1 and the C‐terminus of OsSWEET11b, OsSWEET13, OsSWEET14 and OsSWEET15 were validated via pull‐down (Figure 6b; Figure S11b) and BiFC (Figure 6c). Additionally, we selected OsSWEET15 and ZmXa13 and performed a co‐IP experiment to determine their interaction with OsHMGB1‐HA (Figure 6d). Although we failed to validate the interactions of OsHMGB1 with the complete OsSWEET protein through Y2H and pull‐down assays, we validated that OsHMGB1 could interact with the complete OsSWEET11a/Xa13, OsSWEET11b, OsSWEET13, OsSWEET14 and OsSWEET15 proteins via BiFC experiments (Figure S11c; Figure S12). Interestingly, based on the YFP fluorescence signals, the interactions with the C‐terminus of the OsSWEETs were mostly localized to the plasma membrane and cytoplasm (Figure 6c). However, the interactions with full‐length OsSWEETs were clearly localized on the plasma membrane (Figure S11c; Figure S12). Taken together, the data indicate that OsHMGB1 can interact with the C‐terminus of OsSWEET11a, OsSWEET11b, OsSWEET13‐OsSWEET15 and ZmXa13. These data partially explain the expanded resistance spectrum of oshmgb1 and the conserved function of clade III OsSWEETs.

Figure 6.

OsHMGB1 interacts with the C‐terminus of clade III OsSWEETs. (a, b) The interactions between OsHMGB1 and clade III SWEET proteins were validated by Y2H (a) and pull‐down (b) assays. OsHMGB1 was C‐terminal labelled with a 6 × His tag, and the C‐termini of the OsSWEETs fragments and ZmXa13 were labelled with a 6 × His tag and a GST tag. Pull‐down was performed with GSH magnetic beads. (c) A BiFC assay was used to evaluate the interactions between OsHMGB1 and clade III OsSWEETs. Scale bars, 50 μm. (d) The interactions between OsHMGB1 and the OsSWEET15 or ZmXa13 protein were verified through an in vivo Co‐IP assay.

OsHsp20L interacts with clade III OsSWEETs and negatively regulates bacterial blight resistance

Because the interactions of OsHMGB1 fail to explain the susceptibility of OsSWEET12, we checked the interaction relationship between the clade III OsSWEETs and the second candidate Xa13‐interacting gene, OsHsp20L. First, we determined that OsSWEET11a/Xa13 interacts with OsHsp20L via Y2H (Figure 7a) and co‐IP (Figure 7b). Furthermore, we surprisingly found that all six C‐terminal regions of clade III OsSWEETs interact with OsHsp20L via pull‐down (Figure 7c; Figure S11b) and BiFC (Figure 7d). As expected, the C‐terminus of ZmXa13, but not that of OsSWEET16, could interact with OsHsp20L (Figure 7c,d). Additionally, OsHSP20L could interact with all six full‐length OsSWEETs proteins in BiFC (Figures S11c, S12). Interestingly, we performed interaction assays between OsHsp20L and each of the truncated constructs of OsSWEET11a/Xa13, as shown in Figure S4. In addition to OsHMGB1, OsHsp20L interacts with Xa13^230‐307, Xa13^173‐255 and Xa13^173‐274 (Figure S4).

Figure 7.

OsHsp20L interacts with five clade III OsSWEET proteins. (a, b) Identification of the interaction between OsSWEET11a/Xa13 and OsHsp20L through Y2H (a) and co‐IP (b) assays. (c, d) Physical interactions between OsHsp20L and clade III SWEETs proteins were assessed through pull‐down (c) and BiFC (d) assays. OsHsp20L was labelled with an MBP tag, which was subsequently pulled down with MBP magnetic beads, and immunoblotting was performed with an anti‐GST antibody. Scale bars, 50 μm.

To determine whether OsHsp20L is involved in BB resistance, we generated the RNAi, overexpression and knockout lines of OsHsp20L in ZH11 via CRISPR. Like our findings for OsHMGB1, the susceptibility of the OsHsp20L‐overexpressing lines increased slightly (Table S1, Figure S13a,b), while both the RNAi and knockout lines showed dramatically enhanced resistance to PXO99 (Figure 8a,b; Table S1, Figure S13a,b) in both the T₁ and T₂ generations. In the resistant IRBB13 background, the lesion length and bacterial population increased slightly in the overexpression lines but were not clearly different in the RNAi lines compared to those in the IRBB13 background in both the T₁ (Table S2) and T₂ (Figure S14a,b) generations. Similarly, the OsHsp20L knockout lines exhibited enhanced resistance not only to PXO99 but also to PXO86, suggesting that the spectrum of these genes is broader than that of xa13‐mediated BB resistance. Overall, we concluded that OsHsp20L is involved in clade III OsSWEET‐mediated susceptibility and negatively regulates rice BB resistance.

Figure 8.

OsHMGB1 and OsHsp20L coordinately participate in clade III OsSWEETs‐mediated susceptibility to bacterial blight in rice. Disease symptoms (a) and lesion lengths (b) caused by PXO99 and PXO86; graphic of rice spike (c) and seed setting rate (d) in OsHsp20L knockout lines (hsp20l‐7 and hsp20l‐14), OsHMGB1 knockout lines (hmgb1‐1 and hmgb1‐3) and double knockout lines of hsp20l/hmgb1‐19 and hsp20l/hmgb1‐23. The asterisks above the columns represent the significance difference between wild‐type ZH11 and the transgenic lines (*P ≤ 0.05). (e) Working model of the mechanism by which OsHMGB1 and OsHsp20L coordinately regulate rice BB susceptibility via clade III OsSWEET members.

Oshmgb1 oshsp20l double knockout plants exhibited slightly greater resistance to PXO99 and PXO86 than oshmgb1 and oshsp20l (Figure 8a,b). However, the lesion lengths were still longer than those of ossweet11a for PXO99 and ossweet14 for PXO86 in the Nipponbare and ZH11 backgrounds, respectively. Interestingly, compared with those of ZH11, the seed setting rate of oshsp20l was significantly greater, and the oshmgb1 and oshmgb1 oshsp20l plants were not affected (Figure 8c,d).

Discussion

New functions of OsSWEET11a/Xa13

Since the cloning of the first clade III OsSWEET protein, OsSWEET11a/Xa13, involved in recessive resistance to bacterial blight (Chu et al., 2006; Yang et al., 2006), the function of OsSWEET11a/Xa13 has been extensively studied. As a seven‐transmembrane protein, OsSWEET11a/Xa13 and several clade III members have also been shown to be involved in rice pollen fertility, seed filling and sheath blight resistance (Chu et al., 2006; Fei et al., 2021; Gao et al., 2018; Kim et al., 2019; Wu et al., 2022a; Yang et al., 2018). Both of these subtypes share some common and differential characteristics. For instance, all members of the Xoo strain are susceptible to bacterial blight, which is targeted and activated by cognate TALEs or aTALEs (Zhang et al., 2022). Additionally, OsSWEET11a/Xa13 and OsSWEET11b are involved in male fertility (Chu et al., 2006; Wu et al., 2022a). OsSWEET11a/Xa13, OsSWEET14 and OsSWEET15 play the same roles in seed filling (Fei et al., 2021; Hu et al., 2023; Yang et al., 2018). However, OsSWEET11a/Xa13 and OsSWEET14 play opposite roles in sheath blight resistance (Gao et al., 2018; Kim et al., 2020). Biochemically, OsSWEET11a/Xa13 commonly mediates the transport of sugars (Chen et al., 2010) but is specifically involved in copper uptake (Yuan et al., 2010). In this study, we revealed that the C‐terminus of OsSWEET11a/Xa13 is required for full susceptibility, but it can be replaced with the C‐terminus of ZmXa13. Indeed, OsSWEET11a/Xa13 could be replaced with any other gene, from OsSWEET12 to OsSWEET15, under its native promoter. However, we observed that all the chimeric proteins constructed from cDNA did not fully rescue the susceptibility of IRBB13 cells to infection, as previously reported for pOsSWEET11a‐OsSWEET11a (CDS) (Yuan et al., 2009). These findings suggested that the introns and 5′‐UTR sequences of OsSWEETs may also be important for cell type‐specific expression, as reported for AtSWEET11 (Zhang et al., 2021). Importantly, we revealed two negative immune regulators, OsHMGB1 and OsHsp20L, which interact with most clade III OsSWEETs and mediate direct interference in the rice immune response. Overall, we revealed a novel conserved susceptibility mechanism of clade III OsSWEETs in addition to sugar efflux. In conclusion, we propose that different Xoo strains carry certain TALEs that are secreted into rice cells and activate the expression of cognate OsSWEET proteins, which interact with OsHMGB1 and OsHsp20L to suppress the rice immune response (Figure 8e).

OsHMGB1 and OsHsp20L partially contribute to BB susceptibility independent of sugar efflux

Previously, knockouts of either OsSWEET11a/Xa13 or OsSWEET14/Xa41 were shown to confer full resistance to the PthXo1‐carried strain PXO99 and the AvrXa7‐carried strain PXO86, respectively (Kim et al., 2019; Zeng et al., 2020). Here, we found that both oshmgb1 and oshsp20l significantly attenuated the length of lesions affected by PXO99 and PXO86 (Figure 8a,b). However, the length of these lesions is still shorter than that of lesions identified in ossweet11a or ossweet14 (Kim et al., 2019; Zeng et al., 2020). These findings indicate that OsHMGB1 and OsHsp20L only partially but not completely contribute to OsSWEET‐mediated BB susceptibility (Figures 4 and 8). The oshmgb1 oshsp20l double mutant had a slightly shorter lesion length than the oshsp20l double mutant, which was also longer than that identified in ossweet11a or ossweet14. Furthermore, OsHsp20L interacts with more OsSWEETs than with OsHMGB1. Consistently, oshsp20l was more resistant to PXO99 and PXO86 than oshmgb1 was, as indicated by the shorter lesion length (Figure 8a,b). Taken together, these findings suggested that OsHsp20L and OsHMGB1 coordinately mediate susceptibility to clade III OsSWEETs and synergistically attenuate rice immunity. However, both OsHMGB1 and OsHsp20L interact with the C‐terminus of OsSWEETs. For example, a putative motif of OsSWEET11a/Xa13 ranging from amino acid 230 to 255 was found to interact with OsHMGB1 and OsHsp20L (Figure S4). These data suggested that OsHMGB1 and OsHsp20L may competitively interact with OsSWEETs.

Clade III OsSWEETs are known to transport sugars, hexanose and GA, which are commonly required for bacterial proliferation. The mammalian HMGB1 protein is ordinarily regarded as a DAMP and transcriptional regulator that triggers inflammatory or immune responses and mediates epigenetic regulation. AtHMGB3 was identified as a DAMP molecule that positively triggers plant immunity (Choi et al., 2016). In contrast, knockout or knockdown of OsHMGB1 enhanced rice resistance to BB, suggesting that OsHMGB1 negatively regulates rice immunity rather than functioning as a DAMP molecule to elicit an immune response. Given that OsHMGB1 has DNA binding functions (Wu et al., 2003) and that OsHMGB1 is subcellularly localized to the nucleus (Wang et al., 2023), we believe that the interaction between the C‐terminus of clade III OsSWEETs and OsHMGB1 negatively regulates rice immunity by epigenetic transcription regulation (Wang et al., 2023). Similarly, sHSPs are usually considered to function as molecular chaperones and to positively regulate plant immunity (Ju et al., 2017; Wu et al., 2022b). In our study, we showed that OsHsp20L interacts with all six C‐terminal regions of clade III OsSWEETs and negatively regulates rice BB resistance. As previously reported, ossweet11a, ossweet11b and ossweet15 significantly reduce the seed‐filling rate (Wu et al., 2022a; Yang et al., 2018), and ossweet11a ossweet14 exacerbate more severe phenotypes than does ossweet11a (Fei et al., 2021). Furthermore, as OsSWEET11b did not mediate detectable GA transport, the reduced seed filling rate in ossweet11b suggested that GA was excluded from the general roles of seed filling and susceptibility, leaving sugar efflux alone for the mechanism of OsSWEET11b (Wu et al., 2022a). To our surprise, the singleton oshmgb1 and oshsp20l mutants and the double knockout oshmgb1 and oshsp20l mutants did not exhibit any detectable phenotypic differences in terms of seed filling compared with that of the wild‐type ZH11. The key sugar transport sites of OsSWEET11a were also excluded from the possible motif determining the interaction with OsHMGB1 or OsHsp20L (Chen et al., 2010; Figure S4). Overall, we conclude that OsHMGB1‐ or OsHsp20L‐mediated susceptibility directly suppresses rice immunity independent of sugar efflux.

OsHMGB1 and OsHsp20L are potentially modified after pathogen inoculation

Previously, knockout or knockdown of OsSWEET11a/Xa13 and OsSWEET14/Xa41 was shown to result in significantly enhanced resistance in Xoo strains (Kim et al., 2019; Zeng et al., 2020). However, constitutive expression of allelic OsSWEET11a/Xa13 did not influence resistance in IRBB13 but negatively affected growth (Gao et al., 2018; Yuan et al., 2009). Furthermore, constitutive expression of two interactors of OsSWEET11a/Xa13, COPT1 and COPT5, did not significantly enhance susceptibility in ZH11 or IRBB13 (Yuan et al., 2010). Here, we found that disrupted OsHMGB1 or OsHsp20L could significantly enhance the resistance of four Xoo strains, namely, PXO99, PXO86, PXO61 and YN24. Similarly, constitutive expression of OsHMGB1 or OsHsp20L in ZH11 and IRBB13 increased only a small lesion length and had a slight effect on the mRNA or protein content. Upon pathogen inoculation, the above genes tended to be activated. All these results suggest that the activity of these proteins needs to be modified. This hypothesis is consistent with the subcellular location of the plasma membrane protein OsSWEET11a/Xa13 and its three interactors. OsHsp20L, OsAPX8 and OsHMGB1 are located mainly in the cytoplasm, chloroplast and nucleus, respectively. At least one of the other two genes, OsSWEET11a/Xa13 or its interactor, is required for movement, which is ordinarily mediated by protein modification. Indeed, plant HMGB proteins have been shown to be mobile after phosphorylation by the protein kinase CK2alpha (Choi et al., 2016; Stemmer et al., 2002).

OsHMGB1 and OsHsp20L are candidates for breeding broad‐spectrum BB‐resistant rice

Bacterial blight is an old disease that is prevalent in rice planting areas and is becoming a new danger of outbreaks in China (Xu et al., 2022). Many R genes have been introduced into cultivar varieties to manage BB diseases in practice. However, with the rapid evolution of Xoo strains and lack of disease resistance, even the newly cloned and applied broad‐spectrum R gene of Xa23 has been defeated by emerging isolates (Chen et al., 2022). Recently, CRISPR/Cas9‐mediated gene editing has been broadly used to disrupt OsSWEET11a/Xa13, OsSWEET14/Xa41 and/or Xa5 (Gupta et al., 2023; Kim et al., 2019; Zeng et al., 2020) or to disrupt TALE‐binding elements (EBEs) of OsSWEET11a/Xa13, OsSWEET13/Xa25 and OsSWEET14/Xa41 (Li et al., 2020; Oliva et al., 2019; Xu et al., 2019), which can generate either race‐specific or broad‐spectrum BB‐resistant rice. Alternatively, knocking in the EBE sequence to the dysfunctional executor gene xa23 also results in broad‐spectrum resistance to BB (Gupta et al., 2023). However, direct disruption of OsSWEET11a/Xa13 is often accompanied by dramatic reductions in seed setting and seed filling (Fei et al., 2021; Gao et al., 2018). In this study, we showed that disruption of OsHMGB1 and OsHsp20L via CRISPR/Cas9‐mediated gene editing could also result in moderate and broad‐spectrum resistance to BB without adverse effects on seed setting rates. These findings could lead to good potential candidate resources for breeding BB‐resistant rice in the future.

Methods

Plant materials and growth conditions

The japonica rice variety Zhonghua11 (ZH11, OsSWEET13/xa25), the indica rice variety IR24 (OsSWEET11a/Xa13) and the rice variety IRBB13 (OsSWEET11a/xa13) were used in this study. Rice plants were grown in a plant growth chamber at an appropriate temperature of 28 ± 2°C, a photoperiod of 16 h and a relative humidity of 85–100% or were grown at Huashan Rice Experiment Station, Wuhan University, Hubei Province, China (longitude: 30°55′, latitude: 114°53′), for field experiments as previously reported (Wu et al., 2022c). Nicotiana benthamiana plants were grown at 23 ± 2 °C for 4 weeks and subjected to Agrobacterium‐mediated transient expression.

Vector construction and rice transformation

The primers used in this study are listed in Table S3. The ORFs of the ZmXa13 and clade III OsSWEET genes were amplified and cloned and inserted into the pCAMBIA1300 vector following the promoter sequence of OsSWEET11a/Xa13 (1267 bp upstream sequence from the start codon; reference DQ421396). C‐terminus truncation (N‐terminus of OsSWEET11a/Xa13^1‐220, NR) and domain switching were performed with fusion PCR to generate recombinant N‐termini of OsSWEET11a/Xa13 and C‐terminus of ZmXa13 (ONR) and the N‐terminus of ZmXa13 and C‐terminus of OsSWEET11a/Xa13^221‐307 (ZNR). The full‐length cDNAs of OsHMGB1 (621 bp) and OsHsp20L (777 bp) were isolated from ZH11 via RT–PCR and introduced into the pCXUN vector, which contains the maize Ubiquitin 1 promotor (Chen et al., 2009). The 372‐bp and 279‐bp fragments of OsHMGB1 and OsHsp20L were inversely and repeatedly cloned and inserted into pDS1301 to generate RNAi constructs as previously described (Li et al., 2013). CRISPR/Cas9‐mediated gene‐editing vectors were constructed for OsHMGB1 and/or OsHsp20L according to previous reports (Ma et al., 2015). Transgenic experiments were performed with Agrobacterium tumefaciens‐mediated transformation of mature seeds from the indica IRBB13 or japonica ZH11 background as previously reported (Ge et al., 2006; Li et al., 2013).

Pathogen infection assays

The Philippine Xoo strains PXO99 (PthXo1), PXO61 (PthXo3) and PXO86 (avrXa7) and the Chinese Xoo strain YN24 (PthXo1) were incubated on PSA (10 g/L polypeptone, 1 g/L glutamic acid, 10 g/L sucrose and 15 g/L agar, pH 7.0) plates at 28 °C for 2–3 days and then suspended in 10 mM magnesium chloride (MgCl₂) to an OD₆₀₀ = 0.5. The bacterial suspension was inoculated using the leaf‐clipping method at the booting stage, and the lesion length was measured at 14 or 21 dpi (Chu et al., 2006).

Quantification of gene expression

Total RNA was isolated from the rice leaves using a Plant RNA Kit (Omega Biotek, Norcross). cDNA was synthesized using HiFiScript gDNA Removal RT MasterMix (CWBIO, Beijing, China). Quantitative real‐time PCR was performed on a CFX Connect Real‐Time PCR system (Bio‐Rad) using MagicSYBR Mixture (CWBIO, Beijing). The expression level of OsACTIN was used as the internal control to normalize the results (Ju et al., 2017). Each qRT–PCR assay was repeated at least twice, with three replicates. All the qRT–PCR primers used are listed in Table S3.

In vitro pull‐down assays

The full‐length cDNAs of OsHMGB1 and OsHsp20L were cloned and inserted into Gateway pET‐DEST42 (Invitrogen) and pMAL‐C2X (New England Biolabs) for in vitro expression of the recombinant protein with a C‐terminal 6 × His and N‐terminal MBP tag, respectively. The C‐termini of the OsSWEETs and ZmXa13 were cloned and inserted into pET‐60‐DEST (Novagen, UK) for fusion with a C‐terminal 6 × His tag and an N‐terminal GST tag. All the constructs were separately transformed into Escherichia coli BL21 (DE3) cells. The recombinant proteins were induced with 0.3 to 1 mM IPTG and harvested through ultrasonication and centrifugation. The crude proteins of all the GST‐SWEETs‐His were separately mixed with OsHMGB1‐His or MBP‐OsHsp20L and incubated for more than 6 h in an ice bath. A pull‐down assay was subsequently performed with either the Mag‐Beads GST Fusion Protein Purification Kit (Sangon Biotech, Shanghai, China) or the pMAL™ of the Protein Fusion and Purification System (NEB). The pulled‐down proteins were separated by SDS–PAGE and detected by immunoblotting with an anti‐His or anti‐GST antibody (ABclonal, Wuhan, China) as described previously (Wu et al., 2022c).

In vivo BiFC and Co‐IP analysis

The C‐terminus or full‐length region of OsSWEETs and ZmXa13 and the full‐length cDNA of OsHMGB1 or OsHsp20L were separately cloned and inserted into the pSPYNE and pSPYCE vectors, respectively, for the split‐YFP assay (Walter et al., 2004). The above A. tumefaciens GV3101 plants harbouring recombinant pSPYNE or pSPYCE were mixed at a 1:1 ratio and subsequently coinfiltrated into N. benthamiana cells for transient expression. The YFP signals were observed and photographed with an SP8 laser scanning confocal microscope (Leica, Germany) at 2 or 3 days after infiltration (dpi).

For coimmunoprecipitation (co‐IP) assays, OsHMGB1 or OsHsp20L fused with an HA tag in pSPYCE and a C‐terminal fragment of OsSWEETs and ZmXa13 fused with a myc tag in pSPYNE were transiently coexpressed in N. benthamiana leaves. Total proteins were isolated from leaves at 3 dpi using 3 volumes of protein extraction buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 50 mM sucrose, 0.5% Triton X‐100, 0.5 mM DTT, 10 mM NaF, 1 mM Na₃VO₄, 25 mM β‐glycerolphosphate, 1 mM PMSF and 1× Plant Protease Inhibitor Cocktail) as previously described (Yuan et al., 2010). After the proteins were filtered through a 0.22 μm Miracloth (Merck‐Millipore, Darmstadt, Germany) followed by centrifugation at 15 000  g for 30 min at 4 °C, the supernatant was subjected to immunoprecipitation assays with a Pierce HA Tag Co‐IP Kit (Thermo Scientific Pierce, Rockford). The proteins were captured by anti‐HA magnetic beads and detected by immunoblotting with an anti‐myc antibody (ABclonal). Images were taken with a chemiluminescence imaging system (Tanon, Shanghai, China) or Touch Imager (eBLOT, Shanghai, China).

Yeast two‐hybrid assay

To test protein–protein interactions in yeast cells, baits containing the C‐terminus of OsSWEET clade III were cloned and inserted into pGBKT7, and preys of OsHMGB1 and OsHSP20L were cloned and inserted into pGADT7. Yeast strain Y2HGold cells were transformed with the pGBKT7 and pGADT7 constructs. Positive transformants were selected on synthetic dextrose media lacking leucine and tryptophan (SD/−Trp‐Leu) (Clontech). Protein interaction validation was performed on SD/−Trp‐Leu‐His‐Ade medium supplemented with aureobasidin A.

Phylogenetic analysis

The clade III SWEETs in rice, maize and Arabidopsis were obtained from the EnsemblPlants website (https://plants.ensembl.org). The alignment of amino acid sequences and the phylogenetic tree were constructed using MEGA11 software with the neighbour‐joining method (Tamura et al., 2021). Motif comparisons were obtained from the MEME Suite website (https://meme‐suite.org/meme/index.html).

Conflict of interest

These authors declare no competing interests.

Author contributions

T.W., M.Y. and Z.C. designed the research. X.W., Y.J., T.W. and L.K. performed the experiment. X.W., Y.J., H.L. and X.C. analysed the data. T.W., Y.J., X.W. and Z.C. wrote the paper.

Supporting information

Figure S1 Analysis of clade III OsSWEETs. (a) Phylogenetic analysis of clade III SWEETs in rice, maize and Arabidopsis. (b) Structure of the motifs in clade III OsSWEETs proteins. (c) Amino acid alignment of carboxyl‐terminal of the clade III OsSWEETs.

Figure S2 C‐terminus truncated OsSWEET11a/Xa13 but not ZmXa13 recovered the copper transport ability. (a) The physical interactions between COPT5 and MtN3.1 motif of OsSWEET11a/Xa13 or ZmXa13 using yeast two‐hybrid (Y2H) assay. The interactions were validated by quadruple dropout supplements (QDO) with aureobasidin A (AbA) and 5‐Bromo‐4‐chloro‐3‐indolyl‐alpha‐D‐galactopyranoside (X‐α‐gal). (b) C‐terminus truncated OsSWEET11a/Xa13 recover the copper transport function with COPT1 and COPT5 in ctr1▵ctr3▵ yeast strain. (c) The diagram of vector construction of N‐terminal and C‐termini switch of OsSWEET11a/Xa13 and ZmXa13 genes used for transgenic manipulation.

Figure S3 Validation of the disease resistance in the T₁ generation of C‐terminus truncated, domain switched of OsSWEET11a/Xa13 and ZmXa13 transgenic rice. (a–d) Lesion lengths caused by PXO99 in three individual lines of IRBB13 background at T₁ generation. ZmXa13 (ZR1, ZR2 and ZR14) (a), OsSWEET11a/Xa13 C‐terminus truncated of NR (NR9, NR13 and NR17) (b), chimeric lines of ONR (ONR1, ONR12 and ONR14) containing the N‐terminal of OsSWEET11a/Xa13 and C‐terminus of ZmXa13 (c), chimeric lines of ZNR (ZNR3, ZNR9 and ZNR11) containing the N‐terminal of ZmXa13 and C‐terminus of OsSWEET11a/Xa13 (d). The asterisks upon columns represent the significance between wild‐type IRBB13 and transgenic lines. (*P ≤ 0.05; **P ≤ 0.01).

Figure S4 The putative motif of OsSWEET11a/Xa13 correspond to the interaction with OsHMGB1 and OsHsp20L. Identification of physical interactions of OsHMGB1 (a) and OsHsp20L (b) with C‐terminus of OsSWEET11/Xa13 through Y2H assay. Physical interactions were validated by triple dropout supplements (TDO) with AbA and QDO.

Figure S5 Identification of the function of OsHMGB1 to bacterial blight resistance at T₀ generation. (a, b) Lesion lengths caused by PXO99 (upper) and relative expression level of OsHMGB1 (lower) in T₀ generation of OsHMGB1 suppression lines (a) and overexpression lines (b) of ZH11 background. The asterisks upon columns represent the significance between wild‐type ZH11 and OsHMGB1 transgenic lines (*P ≤ 0.05; **P ≤ 0.01).

Figure S6 Validation of the disease resistance to bacterial blight in T₁ generation of OsHMGB1 suppression and overexpression lines. (a, b) Lesion lengths of OsHMGB1 suppression lines (RNAi‐14, RNAi‐23 and RNAi‐33) (a) and overexpression lines (OE‐15, OE‐16 and OE‐24) (b) caused by PXO99 in T₁ generation of ZH11 background. The asterisks upon columns represent the significance between wild‐type ZH11 and OsHMGB1 transgenic lines. (*P ≤ 0.05; **P ≤ 0.01).

Figure S7 Overexpression of OsHMGB1 increased the susceptibility to PXO99 at T₁ generation in IRBB13 background. Lesion lengths of each of three OsHMGB1 suppression lines (RNAi‐7, RNAi‐8 and RNAi‐9) (a) and overexpression lines (OE‐31, OE‐37 and OE‐38) (b) at T₁ generation in IRBB13 background. Lesion lengths measured at 14 days post inoculation of PXO99. The asterisks upon columns represent the significance between wild‐type IRBB13 and OsHMGB1 transgenic lines (*P ≤ 0.05; **P ≤ 0.01).

Figure S8 Identification of the knockout lines for OsHMGB1 caused by gene editing. (a) The diagram of OsHMGB1 genome structure for gene editing target site. (b) The blast of OsHMGB1 genome sequences of knockout lines (hb1‐1 and hb1‐5). The red arrows indicate the editing sites in genome.

Figure S9 Suppression of OsHMGB1 increases rice resistance to PXO61. Lesion lengths caused by PXO61 for OsHMGB1 suppression lines (RNAi‐14 and RNAi‐23) were measured at 14 dpi in ZH11 background. The asterisks upon columns represent the significance between wild‐type ZH11 and OsHMGB1 suppression lines (**P ≤ 0.01).

Figure S10 Verification of the disease resistance to PXO99 for each clade III OsSWEETs transgenic rice at T₁ generation. Lesion lengths caused by PXO99 at T₁ generation for each three lines of OsSWEET12 (P12‐3, P12‐9 and P12‐4) (a), OsSWEET13/Xa25 (P13‐4, P13‐5 and P13‐6) (b), OsSWEET14/Xa41 (P14‐1, P14‐2 and P14‐4) (c) and OsSWEET15 (P15‐3, P15‐5 and P15‐9) (d). Lesion lengths were measured at 14 dpi in IRBB13 background. The asterisks upon columns represent the significance between wild‐type IRBB13 and IR24 or OsSWEETs transgenic lines (**P ≤ 0.01).

Figure S11 OsSWEET11b interacts with OsHMGB1 and OsHsp20L. Identification of C‐terminus of OsSWEET11b interacts with OsHMGB1 and OsHsp20L by Y2H (a) and GST pull‐down (b). (c) Full‐length OsSWEET1b interacts with OsHMGB1 and OsHsp20L using BiFC. OsSWEET11b^208–261 indicates the C‐terminus range from 208 to 261 amino acid. Anti‐MBP and anti‐GST are used for the detection of tagged proteins by western blot. Scale bars, 50 μm.

Figure S12 Identification of OsHMGB1 and OsHsp20L interact with full‐length OsSWEETs by BiFC assay in N. benthamiana. Scale bars, 50 μm.

Figure S13 OsHsp20L negatively regulates rice resistance to bacterial blight in ZH11 background. (a) Graphics of disease symptoms caused by PXO99 in OsHsp20L suppression (ZDP5, ZDP8, ZDP10 and ZDP29) and overexpression (ZPP3, ZPP4, ZPP7 and ZPP12) lines at 14 dpi in ZH11 background. (b) Bacterial growth curve of PXO99 in four mixed OsHsp20L suppression (OsHsp20L‐RNAi) and overexpression (OsHsp20L‐OE) lines, respectively. Wild‐type ZH11 used as the control.

Figure S14 Overexpression of OsHsp20L attenuated the xa13‐mediated resistance to PXO99. (a) Disease symptoms caused by PXO99 in OsHsp20L overexpression (BPP2, BPP3 and BPP10) and suppression (BHP4, 5, 6) lines at 14 dpi in IRBB13 background. (b) Bacterial growth curve of PXO99 in OsHsp20L overexpression (IRBB13 P20 OE) and suppression (IRBB13 P20 RNAi) lines.

Table S1 Validation of the disease resistance to bacterial blight in T₁ generation of OsHsp20L suppression and overexpression lines of ZH11.

Table S2 Validation of the disease resistance to bacterial blight in T₁ generation of OsHsp20L overexpression lines of IRBB13.

Table S3 Primers used in this study.

Data availability statement

All data discussed in this study can be found in the manuscript and Supplementary Materials.