CaREM1.4 interacts with CaRIN4 to regulate Ralstonia solanacearum tolerance by triggering cell death in pepper

Abstract Remorins, plant-specific proteins, have a significant role in conferring on plants the ability to adapt to adverse environments. However, the precise function of remorins in resistance to biological stress remains largely unknown. Eighteen CaREM genes were identified in pepper genome sequences based on the C-terminal conserved domain that is specific to remorin proteins in this research. Phylogenetic relations, chromosomal localization, motif, gene structures, and promoter regions of these remorins were analyzed and a remorin gene, CaREM1.4, was cloned for further study. The transcription of CaREM1.4 in pepper was induced by infection with Ralstonia solanacearum. Knocking down CaREM1.4 in pepper using virus-induced gene silencing (VIGS) technologies reduced the resistance of pepper plants to R. solanacearum and downregulated the expression of immunity-associated genes. Conversely, transient overexpression of CaREM1.4 in pepper and Nicotiana benthamiana plants triggered hypersensitive response-mediated cell death and upregulated expression of defense-related genes. In addition, CaRIN4-12, which interacted with CaREM1.4 at the plasma membrane and cell nucleus, was knocked down with VIGS, decreasing the susceptibility of Capsicum annuum to R. solanacearum. Furthermore, CaREM1.4 reduced ROS production by interacting with CaRIN4-12 upon co-injection in pepper. Taken together, our findings suggest that CaREM1.4 may function as a positive regulator of the hypersensitive response, and it interacts with CaRIN4-12, which negatively regulates plant immune responses of pepper to R. solanacearum. Our study provides new evidence for comprehending the molecular regulatory network of plant cell death.


Introduction
Remorins (REMs) are plant-specific proteins that are typically marker proteins of the plasma membrane (PM) in all land plants, including ferns and mosses [1,2]. The remorin protein was first detected in potato (Solanum tuberosum) in 1989, and was named pp34 because of its molecular weight of 34 kDa [3]. The protein was later renamed remorin to demonstrate its ability to associate with the PM [4]. Whole-genome sequencing projects analyzed some plant remorin genes. According to the forecast, there are 2,4,8,11,16,19, and 20 remorin genes in Welwitschia mirabilis, Physcomitrella patens, poplars (Populus spp.), foxtail millet (Setaria italica), Arabidopsis thaliana, rice (Oryza sativa), and wheat (Triticum aestivum) genomes, respectively [1,5,6]. Remorin proteins contain a variable N-terminal and a conserved C-terminal domain, where the N-terminus is primarily responsible for structural and functional differences, while the C-terminus is critical for oligomerization and localization in the PM, including the coiled-coil motif [7]. Based on the different N-terminal domains and phylogenetic tree analysis, REMs were divided into six groups, of which groups 1, 2, and 3 were not phylogenetically distinguished but subdivided by domain characteristics [1].
REMs take part in plant growth, development, responses to abiotic and biotic stresses, signal transduction, and fruit ripening [1,[8][9][10][11]. Studies have revealed that REM proteins enhance plant resistance to bacteria, fungi, and viruses [12][13][14][15]. StREM1.3 binds cell wall-derived galacturonides [16] and interacts with the viral protein TGBp1 [12], impairing the movement of virus X into potato [14]. A. thaliana remorin1.3 (AtREM1.3) is phosphorylated to varying degrees upon treatment with a bacterial elicitor [17,18]. AtREM1.2 has been identified to be a RIN4 interacting protein, which is a negative regulator of plant immunity [19]. Furthermore, MtREM2.2 regulates bacterial infection by interacting with symbiotic receptors after phosphorylation by receptor-like kinase (RLK) [13]. Transgenic maize overexpressing the ZmREM1.3 gene has been reported to show increased resistance to the Puccinia polysora. On the contrary, transgenic plants with ZmREM1.3 gene mutation were more likely to be infected with P. polysora than control plants, suggesting that ZmREM1.3 positively regulates the defense response of maize to P. polysora [20]. Fine mapping and expression and mutation analysis have shown that ZmREM6.3 regulates quantitative resistance of maize to northern leaf blight [21]. MtSYMREM1 is a remorin family protein that interacts with symbionts to regulate pathogen infection during nodulation in Medicago truncatula [13]. Some REM genes play negative regulatory roles in disease resistance. AtREM4.1 interacts with SnRK1 protein, resulting in AtREM4.1 phosphorylation and degradation by the 26S proteasome pathway, and plays a key role in imparting susceptibility to beet roll top virus in A. thaliana [22]. Studies have shown that overexpression of SlREM1.3 in tomato plants enhances susceptibility to Phytophthora infestans [23]. In addition, REMs have been showed to play a significant role in responses to abiotic stresses [1,9,24], such as cold, salt, and drought stress [1,6]. Moreover, SlREM1 positively regulated fruit ripening in SlREM1 overexpression and RNA interference (RNAi) lines [10]. Overexpression of SlREM1.3 in tomato (Solanum lycopersicum) promoted leaf senescence [12].
Programmed cell death (PCD) is a basic biological process that leads to the natural suicide of cells [25,26]. Interaction between plant and microorganism usually causes hypersensitive response (HR)-triggered PCD in plant host cells; this response is correlated with increased resistance to some pathogens [27]. PCD onset in plants is similar to biochemical and morphological traits of animal apoptosis, but there are some differences between the two forms of PCD [28]. Plant PCD is an intricate genetically programmed process. Reactive oxygen species (ROS) have been shown to perform a crucial function in regulating cell death [29], senescence [30], and resistance response in plants [31]. Increased ROS levels affect cellular components and trigger cell necrosis, while ROS at lower levels act as signaling molecules [29]. Cai et al. [32] report that transgenic tomatoes overexpressing SlREM1 showed enhanced susceptibility to Botrytis cinerea. In addition, transient expression of SlREM1 in Nicotiana benthamiana induced PCD. ROS produced by respiratory burst oxidase homolog (RBOH) proteins play vital functions in cell death, particularly in the HR [33,34]. Studies have reported that suppression of N. benthamiana RBOH genes reduced HR-like cell death induced by the protein stimulator INF1 [35]. Similarly, suppression of the OsRBOHA gene is known to significantly reduce HR in rice [36].
RPM1-interacting protein 4 (RIN4) plays an important role in both pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) [37]. Previous studies have suggested that RIN4 functions as a negative regulator of PTI [38]. In Arabidopsis, rin4 mutants show an enhanced defense response, which was contrary to observations in plants overexpressing RIN4 [39]. GmRIN4 genes have been reported to negatively regulate the basal resistance of soybean to Pseudomonas syringae and oomycete pathogens [40]. Moreover, RIN4 can be targeted and modified by several different bacterial effectors and can interact with plant RPM1 (resistance to P. syringae pv. maculicola 1), RPS2 (resistance to P. syringae 2), and some other R proteins [38,41,42]. Recent studies have shown that RIN4 may control exocytosis to confer plant immunity. EXO70 protein promotes callose deposition in the cell wall in the presence of f lg22. RIN4 can promote the transport of EXO70E2 vesicles [43], indicating that RIN4 plays a positive role in the basic defense of plants. Additionally, the results of mass spectrometry suggested that REM protein belongs to the RIN4 protein complex in A. thaliana [19]. In Populus euphratica, PeREM6.5 interacted with PeRIN4 to regulate the activity of PM H + -ATPase [24]. However, despite these advances, REMs and RIN4 members still need to be further studied with regard to regulating the immune response of pepper (Capsicum annuum L.) and Ralstonia solanacearum.
Pepper, a Solanaceae plant with huge agricultural importance, has important economic and edible value, but it is susceptible to infection by R. solanacearum, which can have serious impacts on the quality and yield of pepper. R. solanacearum could cause bacterial wilt disease, which is a serious pathogenic bacterium in Solanaceae crops, including pepper, tomato, and potato [44,45]. Despite the commercial importance of C. annuum, the defense mechanisms of peppers against pathogens are largely unknown. To date, the functional and expression characteristics of very few REM genes in Solanaceae crops are known. However, the exact function of most REMs in pepper remains to be further investigated.
In this study, 18 CaREM genes of pepper were identified based on the C-terminal conserved domain of REM proteins. Phylogenetic analysis, chromosomal localization, motif, gene structure, and promoter of these REMs were investigated. In addition, CaREM1.4, a member of the REM family of peppers, was used as the target to preliminarily analyze the function of CaREM in plant responses to pathogens by analyzing the interaction system of pepper and the R. solanacearum pathogen. The transcription of CaREM1.4 in pepper was induced by R. solanacearum. Knocking down CaREM1.4 expression by virus-induced gene silencing (VIGS) attenuated R. solanacearum infection intensity and reduced the accumulation of ROS, triggering cell death. Moreover, CaRIN4-12, which was an immune negative regulator that interacted with CaREM1.4, was knocked down with VIGS. This enhanced the disease resistance of silenced pepper compared with the control. These results suggested that CaREM1.4 is a positive regulator of HR that interacts with CaRIN4-12, which negatively regulates plant immune responses of pepper to R. solanacearum.

Identification and phylogenetic analysis of the pepper remorin gene family
To identify REM genes from C. annuum, the Ensembl database was searched using the conserved sequence at the C-terminal of REM as a query. Based on the search results, 18 complete CaREM coding sequences were identified. In total, 18 CaREM genes were identified according to their characteristic information, including the gene ID, chromosome location number of the ORF (open reading frame), amino acid length, introns, exons, isoelectric point, and molecular weight and so on ( Table 1). The results showed that predicted protein sizes of the encoded CaREMs ranged from 119 to 571 amino acids and isoelectric points were 5.29-10.03. Multi-sequence alignment of the C-terminal domains of 18 pepper REM proteins showed that they all contain a conserved coiled-coil domain (Supplementary Data Fig. S1). To study the phylogenetic relationships among the proteins belonging to the REM family in pepper, we retrieved the 16 A. thaliana REM members [1], 18 tomato REM proteins, and 19 rice REM members to construct evolutionary trees (Supplementary Data Table S1). The 18 CaREM genes identified above were divided into five groups (groups 1, 3, 4, 5, and 6) (Fig. 1). We assigned identical gene numbers to orthologs of pepper according to the method of Raffaele et al. [1].

Chromosomal position and structure analysis of putative CaREM genes
The 18 CaREM genes of pepper identified here were distributed across 10 of the 12 chromosomes, with different distribution densities on different chromosomes (Supplementary Data Fig. S2). For example, chromosomes 2 and 8 contained three CaREM genes, chromosomes 1, 3, 5, 6, and 10 had two CaREM genes, while chromosomes 11 and 12 had one CaREM gene. The specific position of each REM gene on the chromosome is presented in Table 1.
The exon and intron structures of the CaREMs were analyzed to further understand the evolution of CaREMs (Table 1). Our analysis showed intron number to be conserved and ranged from one to six per gene in pepper. The genes from phylogenetic groups 1 (CaREM1, CaREM1. 3

Motif and promoter analysis of the putative CaREM genes
To estimate the diversity and conservation of the CaREMs, motifs were recognized through the MEME program.

CaREM1.4 transcript increases in pepper leaves exposed to R. solanacearum
It has been reported that multiple SlREM proteins in subgroup I are involved in the plant immune response [32]. Quantitative real-time PCR (qRT-PCR) was used to analyze the transcriptional changes of CaREMs belonging to subgroup I in response to R. solanacearum stress (Supplementary Data Fig. S4). The CaActin was used as an internal reference gene. Data represent the means ± standard deviations from three independent values. * * P < .01, significant difference compared with control by Student's t test.
upregulated express ion of CaREM1.4 after R. solanacearum treatment for further analysis because this expression was more significant. The promoter of CaREM1.4 contains cis-acting elements related to the regulation of the immune response, including the TGACG motif [46,47], ERE-box [48], ABRE-box [48], and W-box [49], suggesting that the gene may be related to the response to pathogen infection. To verify the hypothesis, the transcriptional level of CaREM1.4 in leaves of pepper plants after inoculation with R. solanacearum was determined by qRT-PCR (Fig. 2). Specific primer sequences of qRT-PCR are shown in Supplementary Data Table S4. Compared with the mock treatment, the expression level of CaREM1.4 was significantly increased at 12 and 24 hours post-inoculation (hpi) after pepper leaves were injected with R. solanacearum strain, Px1 (phylotype I).
The results revealed that CaREM1.4 was upregulated in response to the induction of R. solanacearum.

Silencing of CaREM1.4 impairs resistance of pepper to R. solanacearum
The transcriptional level of CaREM1.4 was upregulated by R. solanacearum, indicating that CaREM1.4 may be important in pepper immunity. Therefore, we used the tobacco rattle virus (TRV) VIGS system to further research the role of CaREM1.4 in the pepper-R. solanacearum interaction (Fig. 3) Fig. S6). These results for silencing efficiency indicated that only CaREM1.4 was significantly knocked down. In addition, the increased susceptibility of pepper to the pathogens was manifested in increased R. solanacearum population growth. Compared with the non-silenced plants, the number of colony forming units (CFU) of pepper plants with CaREM1.4 silenced was higher at 5 dpi (Fig. 3D). Transcription levels of pepper defense-associated genes, including CaPR1, CaPO2, CaHIR1, and CaSAR82A, were detected in TRV:CaREM1.4 and TRV:00 pepper plants (Fig. 3E). Upon challenge by R. solanacearum, the transcriptional levels of CaPR1, CaPO2, and CaHIR1 in TRV:CaREM1.4 plants were significantly reduced compared with those in control. These results show that CaREM1.4 performed a positive regulatory function in the pepper-R. solanacearum interaction.

Transient overexpression of CaREM1.4 triggers cell death and upregulates immunity-associated genes in C. annuum and N. benthamiana
To further clarify that CaREM1.4 plays a positive regulatory role in the pepper-R. solanacearum interaction, the CaREM1.4 gene was instantaneously expressed in pepper plants by injecting Agrobacterium tumefaciens containing CaREM1.4-GFP plasmids. Transient CaREM1.4 overexpression stimulated cell death after 4 dpi. However, there were no apparent necrotic phenotypes in control plants (Fig. 4A, a). HR-associated cell death was observed by trypan blue staining, indicating that brief overexpression of CaREM1.4 induced a significant necrosis response in pepper leaves, while only a mild HR-mediated necrosis response was observed in leaves injected with A. tumefaciens GV3101 cells carrying empty vectors (Fig. 4A, b). Moreover, leaves with transient expression of CaREM1.4-GFP were stained with DAB (3,3-diaminobenzidine) to detect H 2 O 2 production, while control leaves were not stained (Fig. 4A, c). CaREM1.4 was successfully expressed in western blotting assay (Fig. 4B) and qRT-PCR (Fig. 4C) experiments. The results showed that CaREM1.4 was successfully expressed. In the meantime, we also measured the ion leakage of leaves expressing CaREM1.4-GFP to analyze the effect of PM injury on cell necrosis, and found that pepper leaves with CaREM1.4-GFP agroinfiltration had more ion leakage at 48 hpi than those with GFP (Fig. 4D). Furthermore, the cytosolic levels of ROS were investigated in pepper leaves. Coincidentally, the H 2 O 2 content of leaves expressing CaREM1.4-GFP increased significantly compared with pepper leaves expressing GFP (Fig. 4E). The transcriptional expression of immune marker genes, including CaPR1, CaHIR1, CaPO2, and CaSAR82A, was examined. These results showed that overexpression of CaREM1.4 could increase the relative expression of these defense-associated genes in plants (Fig. 4F).
CaREM1.4 protein consists of 183 amino acids, including an N-terminal and a conserved C-terminal region (Supplementary Data Fig. S7A). To further identify which domains in CaREM1.4 are necessary for cell death, a set of GFP-tagged CaREM1.4 truncates were constructed: the ORF of 183 amino acids (CaREM1. 4 1-183), the N-terminal region (CaREM1.4-N, , and the C-terminal region (CaREM1.4-C, 70-176), were infiltrated into pepper leaves by agroinfiltration. The phenotype of tobacco leaves was examined at 6 dpi, and the results showed that the C-terminal domain was necessary for cell death (Supplementary Data Fig. S7B). Both the N-terminus and the C-terminus of protein CaREM1.4 could be localized on the cell membrane, and the C-terminus

Interaction of CaREM1.4 and CaRIN4-12
RIN4 negatively regulates plant PTI and ETI responses [50,51]. Previous studies showed that PeREM6.5 interacted with PeRIN4 to regulate the activity of PM H + -ATPase in Populus euphratica, suggesting that CaREM1.4 may interact with RIN4 in pepper. To acquire RIN4 genes from C. annuum, the NCBI database was used for searching as a reference genome. Based on the search results, 13 complete CaRIN4 coding sequences were identified. Thirteen C. annuum RIN4 proteins and 25 A. thaliana RIN4 proteins were used to construct the phylogenetic tree (Supplementary Data Table S1). The 13 CaRIN4s were divided into four groups (groups I, II, IV, and V) (Supplementary Data Fig. S9). Split-luciferase (Split-LUC) assays showed that CaREM1.4 can interact with four members of the CaRIN4 family (Supplementary Data Fig. S10). These members represent different groups of the RIN4 family and contain conserved sequences, and we speculate that CaREM1.4 can interact with RIN4 proteins. To further verify the reliability of interaction, CaRIN4-12, a homologue of PeRIN4, was cloned in C. annuum 'Zhongjiao'. The CaRIN4-12 gene was selected for subsequent interaction and functional verification.
The interaction between CaREM1.4 and CaRIN4-12 was tested by yeast two-hybrid (Y2H), Split-LUC complementation, bimolecular f luorescence complementation (BIFC), and coimmunoprecipitation (Co-IP) assays. We first verified the interaction between CaREM1.4 and CaRIN4-12 by yeast Y2H assays. AH109 strains co-expressing CaREM1.4-pGBKT7 and CaRIN4-12-pGADT7 could grow on yeast defective medium and show βgalactosidase activity (Fig. 5A). The interaction between them was further confirmed by Split-LUC assays. Only the region coinjected with CaREM1.4-cLUC and CaRIN4-12-nLUC had strong luminescent signals. No luminescent signals were found in the region co-expressing CaREM1.4-cLUC and GUS-nLUC (Fig. 5B). CaREM1.4 and CaRIN4-12 coding sequences were inserted into pSPYNE(R)173 and pSPYCE(M) vectors, respectively, and infiltrated into tobacco leaves. The physical association between them was further detected by the BIFC method. The f luorescent signals of the interaction between CaREM1.4 and CaRIN4-12 were detected under the microscope after 48 hpi; they were specifically observed in the PM and nucleus. By contrast, no f luorescent signals were observed in the control when co-expressed with CaREM1.4-nYFP and cYFP (Fig. 5C). To validate the interaction between CaREM1.4 and CaRIN4-12 in plants, we performed Co-IP assays. All proteins were tested in the input group, and CaREM1.4-mCherry was coimmunoprecipitated only when CaRIN4-12 was expressed, but not in the negative control GFP (Fig. 5D). In conclusion, these results suggested that CaREM1.4 interacted with CaRIN4-12 in the cell membrane and nucleus.
REMs are PM-associated proteins found in all embryophytes [9]. Although REM proteins are demonstratively related to the PM, they lack transmembrane domains. The PSORT program predicted that CaREM1.4 was localized in the nucleus and cytoplasm. To clarify the functional location of the interaction between CaREM1.4 and CaRIN4-12, we co-expressed GFP/CaRIN4-12-GFP and CaREM1.4-mCherry in N. benthamiana leaves (Fig. 5E). The results indicated that CaREM1.4 and CaRIN4-12 were co-located on the cell membrane and nucleus.
To further analyze the key domain of CaREM1.4 interaction with CaRIN4-12, Split-LUC and BIFC assays were used to verify the interaction between the C-or N-terminal domain of CaREM1.4 and CaRIN4-12 in N. benthamiana leaves. The results showed that CaRIN4-12 interacts with only the Cterminal domain of CaREM1.4, but not the N-terminal domain (Supplementary Data Fig. S11).

CaRIN4-12 reduced reactive oxygen species and cell death produced by CaREM1.4
Previous experiments have shown that overexpression of CaREM1.4 can cause ROS accumulation and cell death. To further study the mechanism of the interaction between CaREM1.4 and CaRIN4-12 in regulating the plant immune response, we coinjected CaREM1.4-GFP and CaRIN4-12-GFP into pepper leaves. CaREM1.4-GFP + GFP was a positive control and GFP empty vector was a negative control. The results showed that, compared with the positive control, the necrosis phenotype after co-injection was significantly weaker than that after injection alone (Fig. 6A). There was significantly less ion leakage in leaves that were transiently overexpressing CaREM1.4-GFP + CaRIN4-12-GFP compared with CaREM1.4-GFP + GFP leaves 48 hours after agro-infiltration of GV3101 strains (Fig. 6B). Transient overexpression of CaRIN4-12-GFP + GFP leaves showed no significant difference in ion leakage compared with GFP alone. In addition, ROS content was quantified by measuring the accumulation of H 2 O 2 in pepper leaves. The H 2 O 2 content in leaves that co-expressed CaREM1.4-GFP + CaRIN4-12-GFP was obviously lower than that of the control. It was higher than in leaves that were injected with the CaRIN4-12-GFP + GFP or GFP empty vector (Fig. 6C). These results suggest that CaRIN4-12 reduced the ROS and cell death produced by CaREM1.4 significantly.

CaRIN4-12 silencing enhances the resistance of pepper to R. solanacearum
To determine whether R. solanacearum infection can induce the expression of CaRIN4-12, transcriptional levels of CaRIN4-12 after inoculation with R. solanacearum were determined by qRT-PCR ( Supplementary Data Fig. S12). The results revealed that CaRIN4-12 was upregulated in response to the induction of R. solanacearum. In order to further study the function of CaRIN4-12 in the defense response of pepper against R. solanacearum, we conducted a functional loss test on pepper seedlings by performing VIGS on CaRIN4-12. A schematic diagram of CaRIN4-12's VIGS vector construction is shown in Supplementary Data Fig. S5. Compared with the control plants, disease resistance in pepper leaves was enhanced after silencing CaRIN4-12 at 8 dpi (Fig. 7A). Disease index of TRV:CaRIN4-12 and TRV:00 pepper plants was recorded from 4 to 10 days after infection with R. solanacearum (Fig. 7B). The bacterial growth in pepper leaves after CaRIN4-12 gene silencing was remarkably lower than that in the control plants at 5 dpi (Fig. 7C). The qRT-PCR results showed that the relative expression of CaRIN4-12 was significantly reduced in the TRV:CaRIN4-12 plants compared with that in the control plants 0, 48, and 120 hours after infection with R. solanacearum (Fig. 7D). Moreover, we assayed the expression of immune-related genes in CaRIN4-12-knockdown plants (Fig. 7E). Transcript abundances of CaPR1, CaPO2, CaHIR1, and CaSAR82A were markedly higher in the CaRIN4-12-silenced plants than in the control. In addition, after silencing the CaRIN4-12 gene the transcript levels of CaREM1.4 were significantly upregulated (Fig. 7F). However, after the silencing of CaREM1.4 the transcript abundances of the CaRIN4-12 gene were unchanged (Supplementary Data Fig. S13). These results shown that knockdown of CaRIN4-12 may increase the disease resistance of pepper against R. solanacearum and that CaRIN4-12 negatively regulates the expression of CaREM1.4.

Discussion
REMs are specific plant proteins that play significant roles in responses to biotic and abiotic stress factors. However, only a few REMs have been found in plants. Recently available genome sequences allow us to systematically study this gene family in A. thaliana, rice, potato, maize (Zea mays), and wheat. Nevertheless, only a few members of the REM family have been identified in vegetables, and they are especially associated with regulating the response of plants to biotic stress. Therefore, the biological function of REM genes in most vegetables need further study.
In the past, the 16 A. thaliana REM members were generally subdivided into six separate groups owing to the remarkable differences in their N-terminal domains [1]. To further explore the phylogenetic relationships of the C. annuum REM genes, we searched the 16 A. thaliana REM proteins [1], along with 19 and 18 REM members from O. sativa and S. lycopersicum. The 18 CaREM genes were also divided into five subfamilies (1, 3, 4, 5, and 6), which were the same as in A. thaliana. Phylogenetic analysis showed that 11 REMs from foxtail millet could be divided into four subgroups [5]. In wheat, 20 TaREMs were identified and divided into six phylogenetic groups [6]. The phylogenetic similarity between C. annuum and A. thaliana REM proteins suggested that they might have been conserved through evolution. In addition, the evolution of gene families can be further understood by analyzing the exon-intron structure and sequence of conserved regions. Studies found that the intron number of identified REM genes was highly variable. The diverse gene subfamilies and structures of the pepper REMs may ref lect the different functions of these genes in biological and non-biological stress.
The subcellular localization of proteins is crucial for their function. REM proteins are widely recognized as marker proteins in the PM in all terrestrial plants and played a significant role in plantmicrobe interactions [12,32], whereas bioinformatics software predicted that 18 CaREM proteins were mainly localized in the cytoplasm and nucleus. In addition, some wheat REM proteins were located in the cytoplasm [6]. Previous studies demonstrated that the C-terminal region performs a decisive function in the lobule mechanism of the REM protein-specific binding membrane domain [52]. The N-terminal region varies greatly under various biological conditions [53]. In our study, CaREM1.4 was mainly localized to the cell membranes and nuclei. Furthermore, both the N-and C-terminal domains of CaREM1.4 could be localized in the PM, but only the C-terminal domains were necessary for nuclear localization, which showed that CaREM1.4 has various structures and functions. These differences may be connected with motif sequences, suggesting that the localization of CaREMs is complicated and varied. Moreover, some studies have shown that the C-terminal of StREM1.3 is essential for its PM localization [8,54]. In a range of deletion mutants, only SlREM1 of 35 Cterminal amino acids was able to localize to the PM [32]. The specific amino acids in CaREM1.4 required for cell membrane localization remain to be further studied.
Plant cell death is critical for plant growth, evolution, and adaptability to the environment [29,32]. The HR is a form of PCD that responds to pathogen attacks and is involved in resistance to biotrophic and semi-biotrophic pathogens [27,[55][56][57]. ROS play crucial functions in the regulation of PCD [27,58], and can act as positive or negative regulators of PCD, depending on requirements and conditions [58][59][60]. SlREM1 positively regulates PCD, which activates the burst of ROS in N. benthamiana leaves. The amino acids from 173 to 187 of SlREM1 have a crucial function in inducing cell death [32]. In this study, CaREM1.4 was involved in mediating plant cell death, where the C-terminal region of CaREM1.4 was necessary to trigger cell death because of its nuclear localization. The results of BIFC experiments showed that the C-terminal domain of CaREM1.4 and CaRIN4-12 interacted on the cell membrane instead of on the nucleus, indicating that CaRIN4-12 changed the nuclear localization of CaREM1.4 and reduced cell death by interacting with the C-terminal domain of CaREM1.4. As for the specific role of CaREM1.4, C-terminal amino acids in the middle stage of cell death are yet to be further studied. In addition, pepper plants with CaREM1.4 knockout were highly susceptible to R. solanacearum infection; they were also impaired in ROS accumulation and hypersensitive cell necrosis in leaves. In contrast, overexpression of CaREM1.4 in C. annuum and N. benthamiana triggered cell death, H 2 O 2 accumulation, and induction of the expression of immune resistance genes. These results suggest that CaREM1.4 positively regulates plant cell death by activating an oxidative burst.
Previously, it has been proved that PeREM6.5 interacts with PeRIN4 to enhance the enzymatic activity of PM H + -ATPase in P. euphratica [24]. In this study, a homologous gene, CaRIN4-12, of PeRIN4 was cloned in pepper, and the interaction of CaREM1.4 and CaRIN4-12 was tested by Y2H, LUC, BIFC, and Co-IP, which suggests that they might be involved in the interaction between pepper and R. solanacearum. RIN4 is targeted by multiple pathogenic effectors, and consequently guarded by different immune receptors [41]. Previous studies have suggested that RIN4 negatively regulates plant PTI and ETI responses [37,38]. After CaRIN4-12 silencing, the transcript abundance of CaREM1.4 was significantly upregulated. Furthermore, the expression of immuneassociated genes was significantly upregulated. These results suggest that CaRIN4-12 was a negative regulator of C. annuum against R. solanacearum infection, which negatively regulated the expression of CaREM1.4. However, recent studies have shown that RIN4 may control exocytosis to confer plant immunity. For example, EXO70 protein promotes callose deposition in the cell wall in the presence of f lg22, which means RIN4 plays a positive role in plant basal defense [43]. The LUC assay showed that the other four RIN4s in pepper could also interact with CaREM1.4, but whether they play a negative regulatory role in the interaction with CaREM1.4 remains to be further studied.
Previous studies have shown that the defense-related genes play an important role in transgenic plants overexpressing REM genes. For example, the expression levels of defense-related genes in ZmREM1.3-overexpressing plants are significantly higher than those in control plants in response to P. polysora infection [20]. Moreover, transient overexpression of NbREM4 in leaves of N. benthamiana induces expression of immunity-related genes [61]. In this study, we found that defense-related genes were significantly reduced and the expression level of the CaRIN4-12 gene remained unchanged after the silencing of CaREM1.4, suggesting that the silencing of CaREM1.4 weakened the resistance of pepper to R. solanacearum. Meanwhile, co-injection of CaREM1.4 and CaRIN4-12 in pepper led CaRIN4-12 to inhibit CaREM1.4-induced necrosis. CaRIN4-12 interacted with CaREM1.4 and negatively regulated the pepper immune response to R. solanacearum, which was similar to the previous results.
In summary, we ascertained that CaREM1.4 overexpression stimulated cell death in C. annuum and N. benthamiana leaves, and CaREM1.4 knockdown decreased the resistance of pepper leaves to R. solanacearum. In addition, CaREM1.4 could interact with CaRIN4-12 and co-localize at the PM and cell nucleus. Furthermore, the CaRIN4-12 gene inhibited the necrosis induced by the CaREM1.4 gene. Our results suggested that CaREM1.4 played a positive role in the plant ROS burst, cell death, and pathogenesis-related genes inducing resistance, while CaRIN4-12 played a negative regulatory role in these processes (Fig. 8). Our findings provided new evidence for comprehending the molecular regulatory network of plant cell death.

Plant materials and R. solanacearum inoculation
Seeds of tobacco (N. benthamiana) and pepper (C. annuum cultivar 'Zhong Jiao',which exhibited medium resistance to R. solanacearum) were obtained from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (IVF CAAS). The growth conditions of these plant materials were as described in previous studies [62,63]. The highly virulent R. solanacearum strain Px1 (phylotype I) used in this study was isolated from wilted samples of pepper from Shanxi province (China) and obtained from the IVF CAAS. Only one strain was used in this study. It was cultivated according to a method described previously [64,65]. The strains were cultured overnight at 180 rpm, 28 • C, suspended in 10 mM MgCl 2 solution, and diluted to a final concentration of 10 8 CFU/ml (OD 600 = 0.8). For root inoculation, pepper plants at the eight-leaf stage were irrigated with R. solanacearum suspension. For leaf inoculation, the third leaf of pepper was inoculated with R. solanacearum using a 1-ml syringe without a needle, and the mock was inoculated with 10 mM MgCl 2 . Leaves were collected at the indicated time points for further analysis. The disease index (from 0 to 4) was calculated according to the wilting degree of pepper plants, namely 0 (no wilting), 1 (1-25% wilted), 2 (26-50% wilted), 3 (51-75% wilted), and 4 (76-100% wilted or dead) [58].

Vector construction
To construct the vector for the transient expression assay, the ORF of CaREM1.4 or CaRIN4-12 without the termination codon was inserted into the SpeI restriction site of the pGinGFP2 vector with a GFP tag. To construct gene-silencing vectors for VIGS, CaREM1.4 and CaRIN4-12 gene fragments of 200-300 bp were identified by searching for the fragment with the lowest similarity to other pepper gene sequences on the NCBI database, then cloned into the gene-silencing vector pTRV2 (Invitrogen), separately. To construct the vector for the Y2H assay, the CaREM1.4 ORF was constructed into the pGBKT7 and the ORF of CaRIN4-12 was subcloned into pGADT7 with the EcoRI/BamHI sites. To construct the vector for Split-LUC assays, double digestion of nLUC and cLUC plasmids was performed with restriction enzymes BamHI/SalI. The ORF of CaREM1.4 and CaRIN4-12 was constructed into the cLUC and nLUC vector, respectively. To generate constructs for the BIFC assay, CaREM1.4 and CaRIN4-12 were fused to pSPYNE(R)173 and pSPYCE(M) vectors with the BamHI/XhoI sites, respectively [66]. To construct the vector for the Co-IP assay, the ORF sequence of CaREM1.4 was constructed into the SpeI restriction site of pBI121MCS-mCherry vector. The primer sequences for construction of all vectors are listed in Supplementary Data Table S4.

Gene identification and phylogenetic tree analysis
The C. annuum genome and protein sequences were downloaded with default parameters from the Ensembl Plants database [67]. To identify the REM gene family members of pepper plants, the REM gene sequences of A. thaliana and S. lycopersicum were used as query objects and BLASTP was used to perform multi-database search and comparison. REM proteins were confirmed to contain conserved REM domains and divided into different groups according to the description of Raffaele et al. [1]. To identify the RIN4-12 gene family members of pepper plants, the C. annuum UCD10Xv1.1 in the NCBI database was used as a reference genome.
Amino acid sequences of REM proteins from C. annuum, A. thaliana, S. lycopersicum, and O. sativa were collected from the Arabidopsis Information Resource, Sol Genomics Network, Rice Genome Annotation Project, and Ensembl database [67], respectively. Amino acid sequences of RIN4-12 proteins from C. annuum and A. thaliana were collected from the Ensembl database [67] and the Arabidopsis Information Resource. Accession numbers of all REMs and RIN4-12 are listed in Supplementary Data Table S1. ClustalX and MEGA7 software was used for sequence alignment and phylogenetic tree construction [68].

Gene structure and promoter analysis
Genomic sequences and ORFs of CaREMs were acquired from the Ensembl database [67]. The exon and intron structures were identified with Gene Structure Display Server version 2.0 [69]. The MEME program [70] was used to analyze conserved motifs of CaREM genes and DNAMAN software was used to align amino acid sequences. The chromosomal locations of CaREM genes were drawn by BTool. The ExPASy tools were used to predict some biochemical properties, such as isoelectric point and molecular weight. Presumed cis-acting regulatory DNA elements of CaREM genes were identified in the 2.0-kb upstream region preceding the translation start site as described previously [71,72]. The putative stress or hormone-responsive cis-acting regulatory elements in these sequences were analyzed as in previous studies [73].

RNA extraction and qRT-PCR analysis
Total RNA extraction of plants and qRT-PCR analysis were performed as detailed in our previous studies [74,75]; three biological replications of each experiment were prepared. A qPCR System (Bio-Rad, USA) was used to quantify the expression of genes with CaActin (GQ339766) as internal control. Livak and Schmittgen's method was used for data analysis [76]. qRT-PCR primers are listed in Supplementary Data Table S4. The major raw qPCR data are given in Supplementary Data Table S5.

Virus-induced gene silencing assay
A TRV-based VIGS assay was used to transiently silence CaREM1. 4 and CaRIN4-12 in this study. The procedure was performed as previously described [63,74]. To avoid the possible silencing of other homologues, a specific fragment of CaREM1.4 and CaRIN4-12 was amplified from the pepper cDNA library using gene-specific primers (Supplementary Data Table S4) and cloned into the VIGS vector pTRV2. A. tumefaciens strains with pTRV1 vector were respectively mixed with constructs containing pTRV2:CaREM1.4, pTRV2:CaRIN4-12, pTRV2:00, and pTRV2:CaPDS at a 1:1 ratio. The mixed A. tumefaciens cells were infiltrated into 12-day-old pepper plants. qRT-PCR was used to detect the silencing efficiency of plants after R. solanacearum infection.

Determination of R. solanacearum colony-forming units
Pepper leaves injected with R. solanacearum were punched and sampled at 48 hpi with a 1-cm hole punch. Leaves from each plant were punched with six holes and put into a 2-ml centrifuge tube; 400 μl of ddH 2 O was added and the samples were mashed with a sterilized grinding rod. Sterile water (600 μl) was added and the samples were mixed well, followed by dilution to 10 −4 , 10 −5 , and 10 −6 gradients. Finally, 400 μl of each gradient dilution was spread evenly on TTC solid medium and cultured in an incubator at 28 • C. After 2 days, the number of colonies on the plate was counted using ImageJ software (National Institutes of Health). Previous studies report more details on this process [64,65].

Transient expression of CaREM1.4 or CaRIN4-12 in pepper plants
For the transient expression assay, A. tumefaciens suspensions (OD 600 = 0.6) containing the pBinGFP2-CaREM1.4 and pBinGFP2-CaRIN4-12 constructs were injected into pepper leaves, respectively. pBinGFP2 was used as a control. At different time points after infiltration, we harvested the injected pepper leaves for subsequent use.

Histochemical staining, electrolyte leakage and reactive oxygen species accumulation determination
The leaves were histologically stained with DAB for H 2 O 2 measurement and trypan blue staining for cell necrosis assays according to a previously published method [32]. Ionic conductivity was used to quantitatively detect cell death in pepper leaves as described previously [77]. Six leaf disks with a diameter of 1 cm were taken from the injection area of the leaves and, after soaking in 5 ml ddH 2 O for 5 hours, were measured as value A using a conductivity meter (Mettler-Toledo, China). After boiling the centrifuge tube with the leaf disks for 20 minutes, ion conductivity was measured as value B and the ion leakage was calculated as (value A/value B) × 100. H 2 O 2 is one of the important reactive oxygen species. The ROS contents of plant leaves were quantified using Micro Hydrogen Peroxide (H 2 O 2 ) Assay Kit (Solarbio, China) with reference to the instructions.

Protein interaction assays
For the Y2H analysis, pGBKT7-CaREM1.4 and pGADT7-CaRIN4-12 were co-transferred into the AH109 strains using the LiAc (lithium acetate) method. Plasmids pGBKT7-P53/pGADT7-SV40 and pGBKT7-CaREM1.4/pGADT7 were transformed into yeast strains as positive and negative controls, respectively. Yeast colonies grown on SD medium lacking Trp/Leu/His/Ade were identified as positive clones. X-α-gal was used to detect interactions by the development of a blue color. Split-LUC analysis was performed as previously described [78]. The vectors CaREM1.4-nLUC and CaRIN4-12-cLUC were co-injected into tobacco leaves. Luciferin (1 mM; AbMole) was applied to the injected area at 48 hpi and LUC activity was detected by the PlantView100 system. For the BIFC assay, CaREM1.4-nYFP and CaRIN4-12-cYFP constructs containing yellow f luorescent protein (YFP) were co-expressed in N. benthamiana leaves. The control group was a combination of CaREM1.4-nYFP and cYFP. YFP f luorescence signals were imaged using a confocal microscope. For the Co-IP assay, pBI121MCS-CaREM1.4/pBinGFP2-CaRIN4-12 and pBI121MCS-CaREM1.4/pBinGFP2 (as a negative control) were co-expressed in tobacco leaves, separately. After 48 hpi, the injected leaves were clipped and ground, and homogenized in RIPA buffer. The extracts were centrifuged and then the supernatant was aspirated. GFP-TRAP beads were added to the supernatant and incubated for 2 hours. The purified samples were then boiled for western blot analysis. Anti-GFP and anti-mCherry were used to detect proteins.

Statistical analysis
The means ± standard deviations of data were analyzed by Microsoft Excel software and significant differences between control and experimental groups were calculated by Student's t test using GraphPad Prism 8.0.