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. 2021 Oct 31;23(2):188–203. doi: 10.1111/mpp.13153

Different epitopes of Ralstonia solanacearum effector RipAW are recognized by two Nicotiana species and trigger immune responses

Yang Niu 1, Shouyang Fu 1, Gong Chen 1, Huijuan Wang 1, Yisa Wang 1, JinXue Hu 1, Xin Jin 1, Mancang Zhang 1, Mingxia Lu 1, Yizhe He 1, Dongdong Wang 2, Yue Chen 1, Yong Zhang 3,4, Núria S Coll 5, Marc Valls 5,6, Cuizhu Zhao 1,✉, Qin Chen 2,✉, Haibin Lu 1,7,✉
PMCID: PMC8743020  PMID: 34719088

Abstract

Diverse pathogen effectors convergently target conserved components in plant immunity guarded by intracellular nucleotide‐binding domain leucine‐rich repeat receptors (NLRs) and activate effector‐triggered immunity (ETI), often causing cell death. Little is known of the differences underlying ETI in different plants triggered by the same effector. In this study, we demonstrated that effector RipAW triggers ETI on Nicotiana benthamiana and Nicotiana tabacum. Both the first 107 amino acids (N1‐107) and RipAW E3‐ligase activity are required but not sufficient for triggering ETI on N. benthamiana. However, on N. tabacum, the N1‐107 fragment is essential and sufficient for inducing cell death. The first 60 amino acids of the protein are not essential for RipAW‐triggered cell death on either N. benthamiana or N. tabacum. Furthermore, simultaneous mutation of both R75 and R78 disrupts RipAW‐triggered ETI on N. tabacum, but not on N. benthamiana. In addition, N. tabacum recognizes more RipAW orthologs than N. benthamiana. These data showcase the commonalities and specificities of RipAW‐activated ETI in two evolutionally related species, suggesting Nicotiana species have acquired different abilities to perceive RipAW and activate plant defences during plant–pathogen co‐evolution.

Keywords: cell death, E3 ligase, effector, effector‐triggered immunity, Nicotiana, Ralstonia solanacearum, RipAW

Nicotiana species show differential recognition systems for the same pathogen effector.

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1. INTRODUCTION

Through evolution, plants have acquired two layers of immunity that enable them to perceive pathogens and induce plant defences. In the first layer, pattern recognition receptors (PRRs) localized on the plasma membrane directly detect conserved microbe‐associated molecular patterns (MAMPs), resulting in activation of pattern‐triggered immunity (PTI) (Wan et al., 2019). In turn, pathogens use effector proteins, which are delivered into the plant cell cytoplasm, to perturb physiological processes and suppress PTI in order to thrive inside plant hosts. Plants have evolved intracellular resistance (R) proteins to directly or indirectly perceive effectors, triggering effector‐triggered immunity (ETI) (Jones & Dangl, 2006). ETI is often accompanied with a hypersensitive response (HR), a local cell death at the pathogen entry site (Balint‐Kurti, 2019). Most R proteins are nucleotide‐binding domain leucine‐rich repeat receptors (NLRs), whose activation and downstream signalling mechanisms are being intensively studied (Jubic et al., 2019; Zhou & Zhang, 2020).

Ralstonia solanacearum is a soilborne pathogen that causes destructive vascular bacterial wilt disease on plants. The pathogen infects more than 200 plant species, including potato, tomato, tobacco, and many ornamental plants (Genin & Denny, 2012). R. solanacearum is considered one of the most important plant bacterial pathogens worldwide due to its long persistence in the soil, unusually large host range, broad geographical distribution, and unavailable resistant crop varieties (Mansfield et al., 2012). The main virulence determinants of R. solanacearum are the type III secretion system (T3SS) and its translocated effector proteins. The genome of R. solanacearum is estimated to encode about 70 T3SS effectors (Genin & Denny, 2012). To date very few R. solanacearum effectors have been functionally characterized in planta (Landry et al., 2020). For example, AWR5 acts as an inhibitor of the target of rapamycin pathway and enhances plant sensitivity to R. solanacearum (Popa et al., 2016). Simultaneously knocking out seven RipG effector genes decreases R. solanacearum virulence on tomato and Arabidopsis (Angot et al., 2006). The effector PopP2 decreases the DNA affinity of defence‐promoting WRKY transcription factors by acetylation, repressing plant defence in Arabidopsis. The RPS4/RRS1‐R receptor pair mimics PopP2 substrates to capture PopP2 and activate plant defence (Le Roux et al., 2015; Sarris et al., 2015). Effector RipAY degrades glutathione in plant cells through its γ‐glutamyl cyclotransferase (GGCT) activity, altering the intracellular redox environment and suppressing plant immune responses (Fujiwara et al., 2016; Mukaihara et al., 2016; Sang et al., 2018), and RipAC interrupts MAPK‐mediated phosphorylation of SGT1 to suppress ETI (Yu et al., 2020). Some effectors, such as Brg11, RipAK, and RipI, hijack the metabolism to promote R. solanacearum growth in planta (Wang et al., 2021; Wu et al., 2019; Xian et al., 2020).

Protein modification by ubiquitin is one of the most important posttranslational modifications in eukaryotes. It modulates the stability, activity, and sublocalization of target proteins (Kerscher et al., 2006; Zheng & Shabek, 2017). Ubiquitination is accomplished by the sequential function of three enzymes: a ubiquitin‐activating enzyme (E1), a ubiquitin‐conjugating enzyme (E2), and a ubiquitin ligase (E3). E3 ligases catalyse the transfer of ubiquitin from an E2 to lysine residues of the target substrates. E3 ligases have been categorized into the REALLY NEW INTERESTING GENE (RING) family and HOMOLOGUES TO THE E6AP CARBOXYL TERMINUS (HECT) family. RING‐type E3 ligases directly transfer ubiquitin from E2 to their substrates, whereas the HECT‐type E3 ligases form a thioester‐linked intermediate with ubiquitin before ubiquitin attaches to the substrate (Zheng & Shabek, 2017). Albeit ubiquitination does not exist in prokaryotes, effectors from many pathogens mimic E3 ligases to promote their virulence on hosts (Spallek et al., 2009). Examples include Pseudomonas syringae AvrPtoB, XopL from Xanthomonas campestris, and RipG7 effectors from R. solanacearum. AvrPtoB belongs to the RING E3 ligase family and suppresses Fen‐mediated ETI on tomato and PRRs‐mediated PTI in Arabidopsis by ubiquitinating and degrading Fen and PRRs through the 26S proteasome (Abramovitch et al., 2006; Gimenez‐Ibanez et al., 2009; Gohre et al., 2008; Janjusevic et al., 2006). XopL acts as a RING‐finger E3 ligase, whose E3 activity is required to eliminate stromules and induce plastid clustering (Erickson et al., 2018; Singer et al., 2013). RipG7 also harbours E3 ligase activity on still unknown substrates and plays a crucial role in pathogenicity on Arabidopsis, tomato, and Medicago truncatula (Angot et al., 2006). Recently, a conserved cysteine‐dependent E3 ligase domain has been identified in animal and plant bacterial effectors such as SspH2 and SspH1 from Salmonella, IpaH9.8 and IpaH3 from Shigella, and NopM from Rhizobium. They are named as the NEW E3 LIGASE (NEL) family because of their difference in both structure and mechanism from all known E3 ligases (Quezada et al., 2009; Singer et al., 2008; Xin et al., 2012; Zhu et al., 2008). RipAW from R. solanacearum RS1000 was reported to be a new member of the NEL family and to suppress PTI in Nicotiana benthamiana (Nakano et al., 2017). It has also been demonstrated that another R. solanacearum effector, RipV2, contains a NEL domain and plays a critical role in R. solanacearum infection on potato (Chen et al., 2021).

Species of Nicotiana are not only hosts for some R. solanacearum strains but also a very useful model to test gene function by the Agrobacterium‐mediated transient expression assay. This transient expression system is widely used to study R. solanacearum effector function in hosts (Bally et al., 2018; Sabbagh et al., 2019). R. solanacearum effectors RipE1 and RipAW have been shown to subvert the flg22‐triggered reactive oxygen species (ROS) burst, callose deposition, and defence‐related gene expression on N. benthamiana (Jeon et al., 2020; Nakano & Mukaihara, 2019a; Nakano et al., 2017). Other effectors, such as RipB, RipE1, PopP1, and AvrA, are recognized by Nicotiana spp., leading to activation of ETI and cell death (Jeon et al., 2020; Nakano & Mukaihara, 2019b; Poueymiro et al., 2009; Sang et al., 2020; Sun et al., 2020; Zheng et al., 2019; Zhuo et al., 2020). Some R. solanacearum effectors have acquired the ability to repress ETI. For example, RipAK targets catalases and inhibits their activities to abrogate R. solanacearum‐triggered HR on Nicotiana tabacum ‘Xanthi’ (Sun et al., 2017) and RipAY degrades cellular glutathione to inhibit RipE1‐triggered cell death on N. benthamiana (Sang et al., 2020).

Although several R. solanacearum effectors have been shown to trigger cell death in different Nicotiana species (Jeon et al., 2020; Landry et al., 2020), the molecular mechanism underlying this phenomenon has not been deeply investigated. In this study we describe that RipAW, a R. solanacearum GMI1000 effector, uses different ways to trigger cell death in N. benthamiana and N. tabacum, suggesting that different strategies have evolved in a single plant genus to detect the same pathogen effector.

2. RESULTS

2.1. Cell death on N. benthamiana caused by the effector RipAW is NbSGT1‐dependent

R. solanacearum GMI1000 (phylotype I) triggers a very strong HR on N. benthamiana and N. tabacum, while the distantly related strain UW551 (phylotype II) does not trigger this response (Poueymiro et al., 2009). To find out whether other phylotype II strains fail to trigger cell death in both Nicotiana species, bacterial solutions of strains CIP301 and CFBP2957 (phylotype IIA) and UY031, UW551, IPO1609, and NCPPB3987 (phylotype IIB) as well as the control GMI1000 were directly infiltrated into the leaves of Nicotiana spp. Strains GMI1000, CIP301, and CFBP2957, but not those belonging to phylotype IIB (UY031, UW551, IPO1609, and NCPPB3987) induced strong cell death in N. benthamiana and N. tabacum at 3 days postinoculation (dpi; Figure 1a,b). However, cell death trigged by CIP301 in both plants was weaker than that caused by GMI1000 and CFBP2957 (Figure 1a,b). A functional T3SS is required for cell death triggered by the strain GMI1000 on Nicotiana species (Poueymiro et al., 2009), implying that the observed cell death in Figure 1a could be attributed to the activity of an effector. Instead of analysing the natural variation of all known cell death‐triggering effectors among strains (Jeon et al., 2020; Landry et al., 2020; Sang et al., 2020), we preferred to explore new cell death‐triggering effectors. We guessed that the effectors might be conserved among cell death‐triggering strains but absent from those strains unable to cause this response. Because the genomes of CIP301 and NCPPB3987 are not available on public databases, we searched which effectors were present in GMI1000 and CFBP2957 but absent in UW551 and UY031. Using the Ralsto T3E tool (www.ralsto‐T3E.org) (Sabbagh et al., 2019), we identified only seven effectors that met this criterion: RipAW, RipAZ1, RipAU, RipAF1, RipA4, RipS4, and RipJ. To determine whether these effectors triggered cell death, RipAW, RipAZ1, RipAU, and RipAF1 from the strain GMI1000 and green fluorescent protein (GFP) as a control were transiently expressed in N. benthamiana by agroinfiltration. We found that RipAF1, RipAZ1, RipAU, or GFP did not trigger any visible cell death, but RipAW induced a very strong tissue collapse and ion leakage on N. benthamiana (Figures 1c and S1). This observation suggested that RipAW might contribute to GMI1000 and CFBP2957‐induced cell death in Nicotiana spp.

FIGURE 1.

FIGURE 1

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RipAW triggers cell death on Nicotiana benthamiana. Five‐week‐old Nicotiana spp. were leaf‐inoculated with Ralstonia solanacearum strains or agrobacteria containing the effector constructs. (a, b) R. solanacearum strains GMI1000, CFBP2957, and CIP301 trigger cell death in N. benthamiana (a) and Nicotiana tabacum (b). Leaves inoculated with R. solanacearum strains were digitally photographed at 4 days postinoculation (dpi). (c) RipAW, but not RipAU, RipAF1, and RipAZ1, is able to trigger cell death in N. benthamiana. The photographs of leaves expressing those effectors were taken with a camera at 3 dpi. (d) Expression of RipAW enhances accumulation of NbPR1. The total RNA was extracted from the agroinfiltrated N. benthamiana leaves with RipAW and green fluorescent protein (GFP) control at 24 hours postinoculation (hpi). The expression of NbPR‐1 was determined by reverse transcription quantitative PCR and normalized to NbActin. (e) Expression of RipAW limits R. solanacearum JY1 growth. RipAW and control GFP were agroinfiltrated into N. benthamiana leaves. After 24 h, R. solanacearum JY1 strain solution (105 cfu) was infiltrated into the same leaves. Then the JY strain growth in leaves expressing RipAW was measured at the indicated times. Significant differences were evaluated with Student's t test (**p < 0.01). (f) NbSGT1 is required for RipAW‐triggered cell death on N. benthamiana. The first two true leaves of 2‐week‐old N. benthamiana plants were infiltrated with agrobacteria carrying TRV1, TRV2::NbSGT1, TRV2::GFP, and TRV2::NbPDS. Then NbSGT1‐silencing plants were agroinfiltrated with RipAW and investigated for RipAW‐triggered cell death. The picture of RipAW‐triggered cell death on N. benthamiana was taken at 5 dpi. All experiments were repeated three times with similar results. Strain names have been shortened in the image as follows: GMI1000 (GM), CFBP2957 (CF), CIP301 (CP), IPO1609 (IP), UW551 (UW), NCPPB3987 (NC), and UY031 (UY). H represents the number of leaves showing cell death, T represents the total number of R. solanacearum inoculated leaves

To test whether cell death triggered by RipAW results from the activation of plant immune responses, we checked the expression of PATHOGENESIS‐RELATED 1 (PR1) on N. benthamiana, used as a marker gene of salicylic acid‐mediated plant immunity (Ding & Ding, 2020). RipAW expression significantly increased the expression level of NbPR1 compared with that of GFP (Figure 1d). To determine whether or not the immune response triggered by RipAW affects the proliferation of R. solanacearum in plants, we measured R. solanacearum JY1 growth on RipAW‐expressing N. benthamiana leaves. Compared with GFP control, the expression of RipAW significantly restricted JY1 growth (Figure 1e). The expression of GFP and RipAW was detected by western blot (Figure S2). This suggests that RipAW can be recognized by N. benthamiana, activating plant immunity and cell death, which is a typical feature of ETI (Jones & Dangl, 2006). SGT1 plays a critical role in ETI signalling by regulating the stability of NLR proteins (Azevedo et al., 2002; Holt et al., 2005). To examine whether or not RipAW‐triggered cell death is dependent on NbSGT1, we investigated RipAW‐triggered cell death in N. benthamiana after knocking down NbSGT1 by virus‐induced gene silencing (VIGS). We found that the cell death triggered by RipAW was specifically abolished in NbSGT1‐silenced plants, but not in control plants (Figures 1f and S3). This suggests that RipAW might be perceived by (an) NLR receptor(s) eliciting ETI in N. benthamiana.

2.2. E3 ligase activity is required, but not sufficient for RipAW‐triggered cell death in N. benthamiana

RipAW from the strain GMI1000 (RipAWGM) induced strong cell death (Figure 1), while a previous report showed that RipAW from strain RS1000 (RipAWRS) induced leaf chlorosis on N. benthamiana instead of cell death, and that this phenotype was dependent on its E3 ligase activity (Nakano et al., 2017). We decided to test whether RipAWGM‐triggered cell death was caused by a loss of its ligase activity. Recombinant glutathione S‐transferase (GST)‐tagged RipAWGM purified from Escherichia coli was directly incubated with human E1, human UbcH5B (E2), and the substrate HA‐Ubiquitin in vitro, and the reaction mixture was subjected to western blot. A poly‐ubiquitin ladder was detected with anti‐HA antibody (Figure S4) indicating that RipAWGM is a functional E3 ligase. Importantly, the poly‐ubiquitin ladder was completely abrogated when cysteine 177 was substituted by an alanine or a serine (Figure S4), suggesting that this is the key catalytic residue for RipAW E3 ligase activity, consistent with the NEL family features (Nakano et al., 2017; Quezada et al., 2009).

Subsequently, to understand whether the E3 ligase domain is sufficient for RipAW‐triggered cell death, we split the full length RipAW into an N‐terminal fragment spanning 1–107 residues [RipAW (ΔNEL)] and an NEL‐domain containing fragment [RipAW (ΔN1‐107)] (Figure 2a). Neither RipAW (ΔNEL) nor RipAW (ΔN1‐107) caused leaf tissue collapse (Figure 2b). However, electrolyte leakage assay showed RipAW (ΔN1‐107) induced higher conductivity than RipAW (ΔNEL) (Figure 2c). Western blot analysis with anti‐GFP antibody confirmed the expression of RipAW and its variants in N. benthamiana (Figure 2f). These results indicate that both the N‐terminus and the NEL domain are required for RipAW‐triggered cell death in N. benthamiana. To explore the role of the E3 ligase activity in RipAW‐triggered cell death in plants, we analysed cell death in N. benthamiana leaves expressing RipAW (C177A). In contrast to clear tissue collapse caused by RipAW, the C177A mutation completely abolished cell death symptoms (Figure 2b). We further quantified cell death by measuring ion leakage and found that the conductivity caused by RipAW (C177A) was comparable to the GFP control and clearly lower than that caused by RipAW (Figure 2c). These results indicate that the E3 ligase activity of RipAW is essential for cell death in N. benthamiana.

FIGURE 2.

FIGURE 2

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E3 ligase activity is essential for RipAW‐triggered cell death in Nicotiana benthamiana, but not in Nicotiana tabacum. RipAW and its derivatives were transiently expressed in leaves of 5‐week‐old Nicotiana spp. by agroinfiltration. (a) Schematic illustration of RipAW and its derivatives RipAW (ΔNEL), RipAW (ΔN1‐107), and RipAW (C177A). (b) Loss of E3 ligase activity abolishes RipAW‐triggered cell death in N. benthamiana. Cell death phenotype on N. benthamiana was photographed at 4 days postinoculation (dpi) with a digital camera. (c) Cell death in (b) was quantified by investigating electrolyte leakage. The N. benthamiana leaves expressing RipAW and its derivatives were harvested at 4 dpi and the conductivity was measured by a conductimeter. Electrolyte leakage was manifested as the percentage of sample conductivityunboiling/conductivityboiling (the following assay is similar). Different letters above the columns indicate significant differences between conditions (one‐way analysis of variance, Tukey's test, p < 0.01). (d) E3 ligase activity is not required for RipAW‐triggered cell death in N. tabacum. Photographs of cell death in N. tabacum induced by RipAW and its mutants were taken at 4 dpi. (e) Cell death in (d) was quantified by measuring electrolyte leakage. The agroinfiltrated leaves were collected at 4 dpi and their electrolyte leakage was measured with a conductimeter. Asterisks indicate significant differences between RipAW mutants and RipAW (FL) (Student's t test, ****p < 0.001). (f) Detection of RipAW and its derivatives in N. benthamiana with western blot using anti‐GFP antibody. Leaves expressing RipAW and its mutants were harvested and protein was extracted around 24 h after agroinfiltration. H is the number of leaves with cell death and T is the total number of infiltrated leaves. All experiments were repeated three times with the similar results

2.3. The 107 N‐terminal amino acid residues, not its E3 ligase activity, is required for RipAW‐ triggered cell death on N. tabacum

To test whether RipAW and its variants also trigger cell death in N. tabacum, we agroinfiltrated them and investigated cell death display in N. tabacum. When the full length of RipAW was expressed, a very clear cell death phenotype appeared compared to the GFP control, which is consistent with the cell death‐inducing ability of the strain GMI1000 in N. tabacum (Figure 1b). Unexpectedly, RipAW (ΔNEL), but not RipAW (ΔN1‐107), retained the ability to induce cell death in N. tabacum (Figure 2d,e), implying that the first 107 amino acids fragment (N1‐107) of RipAW are required and sufficient for eliciting cell death in N. tabacum. In line with this, the E3 ligase mutant RipAW (C177A) still kept the ability to induce cell death (Figure 2d,e). These results indicate that in contrast to N. benthamiana, the RipAW 107 N‐terminal amino acids fragment, but not E3 ligase activity, is required for RipAW‐triggered cell death in N. tabacum, suggesting that N. tabacum and N. benthamiana employ different strategies to initiate the cell death programme on RipAW recognition.

2.4. The first 60 amino acids of RipAW are not required for the activation of cell death on Nicotiana spp.

The N1‐107 fragment is required for RipAW‐triggered cell death in both N. benthamiana and N. tabacum (Figure 2). To further check the effect of the N‐terminus on the function of RipAW on Nicotiana spp., we generated a series of truncated RipAW versions by deleting 30 amino acids from its N‐terminus and investigated cell death by transiently expressing them in N. benthamiana (Figure 3a). Leaves infiltrated with RipAW (ΔN1‐60) displayed strong cell death phenotypes, while those infiltrated with RipAW (ΔN1‐30) and RipAW (ΔN1‐90) looked healthy (Figure 3b). Consistent with the leaf symptoms, ion leakage assays showed that leaf conductivity triggered by RipAW (ΔN1‐60) was indiscernable from that of RipAW, which was clearly higher than RipAW (ΔN1‐30) and RipAW (ΔN1‐90) (Figure 3c). Similar to N. benthamiana, deletion of these first 60 amino acids did not affect RipAW‐triggered cell death in N. tabacum, but lack of the first 90 amino acids blocked cell death appearance (Figure 3d). Western blot analysis indicated that RipAW (ΔN1‐30) was not expressed in Nicotiana spp., which may underpin the failure of RipAW (ΔN1‐30) to elicit cell death (Figure 3e). Interestingly, all truncated proteins were detected as a single band, while the full‐length RipAW showed two bands (Figure 3e).

FIGURE 3.

FIGURE 3

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The first 60 N‐terminal amino acids are not required for RipAW‐triggered cell death on Nicotiana spp. RipAW N‐terminal deletion mutants were agroinfiltrated into leaves of 5‐week‐old N. benthamiana and N. tabacum. (a) Schematic illustration of RipAW N‐terminal mutants. (b) Deletion of the first 60 amino acids does not affect RipAW‐triggered cell death on N. benthamiana. Leaves agroinfiltrated with RipAW mutants were digitally photographed at 4 days postinoculation (dpi). (c) Cell death triggered by the truncated RipAW variants in (b) was quantified by measuring ion leakage. N. benthamiana leaves expressing the truncated RipAW variants were collected at 4 dpi and immersed into double‐deionized water. Then their ion leakage was measured with a conductimeter. Different letters above the columns indicate significant differences between conditions (one‐way analysis of variance, Tukey's test, p < 0.01). (d) The first 60 amino acids are not essential for RipAW‐triggered N. tabacum cell death. N. tabacum leaves agroinfitrated with the truncated RipAW mutants were photographed at 5 dpi. (e) Detection of RipAW and its mutant variants in N. benthamiana with western blot using anti‐FLAG antibody. The leaves expressing RipAW and its mutants were harvested and protein was extracted around 24 h after agroinfiltration. H is the number of leaves displaying cell death and T is the total number of infiltrated leaves. All experiments were repeated three times with similar results

2.5. RipAW orthologs from different strains exhibit differential abilities to trigger cell death on Nicotiana species

RipAW is present in the genomes of 89 of the 155 sequenced R. solanacearum strains (Sabbagh et al., 2019). To explore if RipAWs from different R. solanacearum strains have acquired the ability to subvert recognition by the immune surveillance system of N. benthamiana, we synthesized RipAW orthologs from representative strains in the four phylotypes: RS1000 (RS, phylotype I), CFBP2957 (CF, phylotype IIA), Po82 (Po, phylotype IIB), and CMR15 (CM, phylotype III) and PSI07 (PS, phylotype IV) (Sabbagh et al., 2019), and expressed these RipAW orthologs (RipAWRS, RipAWCM, RipAWPS, RipAWCF, and RipAWPo) by agroinfiltration on Nicotiana spp. We found that RipAWRS induced clear cell death in leaves of N. benthamiana, while other RipAWs did not trigger this response (Figure 4a). Western blot analysis showed that RipAWCM and RipAWPS were undetectable, therefore they were excluded from subsequent assays (Figure 4c). Consistent with the observed macroscopic cell death, leaves expressing RipAWCF and RipAWPo showed significantly lower conductivities than those expressing RipAWGM and RipAWRS (Figure 4b). This indicates that RipAWCF and RipAWPo might escape perception by N. benthamiana. In contrast, when inoculated on N. tabacum leaves, RipAWPo caused a strong cell death phenotype (Figure 4d). This reinforces the idea that these two Nicotiana species have evolved different surveillance systems to recognize members of the RipAW family.

FIGURE 4.

FIGURE 4

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RipAW orthologs exhibit differential abilities to trigger cell death on Nicotiana spp. RipAW members from different strains were transiently expressed on leaves of 5‐week‐old Nicotiana spp. by agroinifiltration. (a) RipAWRS orthologs triggered strong cell death on N. benthamiana. Leaves expressing RipAW orthologs were photographed at 4 days postinoculation (dpi). (b) Cell death in (a) was quantified by measuring electrolyte leakage. The conductivities of N. benthamiana leaves agroinfiltrated with RipAW orthologs were measured with a conductimeter at 4 dpi. Different letters above columns indicate significant differences between conditions (one‐way analysis of variance, Tukey's test, p < 0.01). (c) The expression of RipAW orthologs in N. benthamiana was detected by western blot using anti‐FLAG antibody. Leaves expressing RipAW orthologs were harvested and protein was extracted at 24 h after agroinfiltration. (d) RipAWRS and RipAWPo triggered N. tabacum cell death. Leaves agroinfiltrated with RipAW family members were photographed at 5 dpi. H is the number of leaves with cell death and T is the total number of infiltrated leaves. The experiments were repeated three times with similar results

2.6. N‐terminus of RipAW orthologs show differential abilities to induce cell death in N. tabacum

The N1‐107 region is responsible for cell death induced by RipAWGM in N. tabacum (Figure 2). Therefore we speculated that natural variation on the N‐termini of allelic RipAWs might enable the RipAW N‐terminus to overcome the N. tabacum recognition system. Because RipAWCM and RipAWPS could not be detected by western blot (Figure 4), we investigated the role in cell death of RipAWGM, RipAWRS, RipAWCF, and RipAWPo N‐termini. We aligned the protein sequences of RipAW orthologs (Figure S5), identified and cloned their N‐termini, and expressed them in N. tabacum by agroinfiltration. Both cell death symptoms and ion leakage assays showed that RipAW (N1‐107)GM, RipAW (N1‐107)RS, and RipAW(N1‐90)CF elicited strong cell death and ion leakage in N. tabacum leaves while RipAW (N1‐90)Po did not cause any cell death in N. tabacum (Figure 5a,b). These data suggest that although N. tabacum possesses a very powerful surveillance system to detect the divergent N‐termini of RipAW orthologs and activate cell death, some R. solanacearum strains have acquired supposedly undetectable variations at RipAW N‐termini as a strategy to overcome this surveillance system during the arms race between R. solanacearum and its plant hosts.

FIGURE 5.

FIGURE 5

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RipAW (N1‐90)CF and RipAW (N1‐107)RS induce strong cell death on Nicotiana tabacum. (a) Expression of RipAW (N1‐90)CF and RipAW (N1‐107)RS caused strong cell death. Leaves transiently expressing the N‐terminus of RipAW orthologs were photographed at 4 days postinoculation (dpi). (b) Cell death in (a) was quantified by testing electrolyte leakage. Leaves agroinfiltrated with the N‐terminus of RipAW orthologs were collected at 4 dpi and immersed in double‐deionized water. Ion leakage was measured with a conductimeter. Different letters above the columns indicate significant differences between conditions (one‐way analysis of variance, Tukey's test, p < 0.01). H is the number of dead leaves and T is the total number of infiltrated leaves. The experiments were repeated three times with similar results

2.7. N‐terminal arginines R75 and R78 are required for RipAWGM‐triggered cell death in N. tabacum, but not in N. benthamiana

To explore how RipAWCF and RipAWPo escape N. benthamina detection, we analysed their protein sequences using Cluster Omega. Strikingly, in comparison to RipAWGM and RipAWRS, RipAWCF and RipAWPo lacked a 17 amino acid fragment after position 61 (Figure S5). This raised the possibility that this N‐terminal peptide may function in RipAWGM‐induced cell death on N. benthamiana. Because charged amino acids normally play very important roles in maintaining protein configurations and in protein–protein interactions, we generated two RipAWGM double point mutants, RipAW (H62D/H64D) and RipAW (R75A/R78A), by substituting two histidine and two arginine residues with aspartate and alanine, respectively, in this region. These mutated versions of RipAW were agroinfiltrated on N. benthamiana and cell death was investigated. While the H62D/H64D mutations did not affect cell death caused by RipAW, the expression of RipAW (R75A/R78A) resulted in patchy yellowing of the leaves, suggesting that the R75A and R78A mutations attenuate RipAW‐triggered cell death in N. benthamiana (Figure 6a). However, quantification of cell death by ion leakage did not show significant differences between RipAW and RipAW (R75A/R78A), although the conductivity value triggered by RipAW (R75A/R78A) was slightly lower (Figure 6b). Both mutated variants of RipAW showed similar protein levels to the wild‐type protein (Figure 6e). These results indicate that the arginine R75/R78 and histidine H62/H64 residues of RipAW have a negligible role in cell death elicited by this effector in N. benthamiana.

FIGURE 6.

FIGURE 6

Open in a new tab

Arginine in positions 75 and 78 of RipAW is essential for cell death in Nicotiana tabacum. The indicated RipAW versions were agroinfiltrated into leaves of 5‐week‐old Nicotiana spp. (a) Substitution of both R75/R78 for alanine and H62/H64 for aspartate did not affect tissue collapse elicited by RipAW in N. benthamiana. Cell death symptoms on leaves expressing RipAW (R75/78A) and RipAW (H62/64D) were photographed at 4 days postinoculation (dpi). (b) Cell death induced by RipAW (R75/78A) and RipAW (H62/64D) on N. benthamiana was quantified by electrolyte leakage. Leaves expressing RipAW (R75/78A) and RipAW (H62/64D) were collected at 4 dpi and immersed in double‐deionized water. The conductivity of samples was measured by a conductimeter. Different letters above columns indicate the significant difference (one‐way analysis of variance, Tukey's test, p < 0.01). (c) Simultaneous mutation on R75 and R78 abolished cell death triggered by RipAW on N. tabacum. Cell death symptoms caused by RipAW (R75/78A) were photographed and the leaves showing cell death were counted at 4 dpi. (d) Cell death in (c) was quantified by electrolyte leakage assay. N. tabacum leaves agroinfiltrated with RipAW and its point mutants were harvested at 4 dpi and put into double‐deionized water. Electrolyte leakage was detected by a conductimeter. Asterisks indicate statistically significant differences between RipAW and its variants (Student's t test, ****p < 0.001). (e) Expression of RipAW and its mutants was detected by western blot with anti‐FLAG antibody in N. benthamiana. Leaves expressing RipAW point mutants were harvested and protein was extracted 24 h after agroinfiltration. H is the number of leaves with cell death and T is the total number of infiltrated leaves. The experiments were repeated three times with the similar results

Considering that the N‐terminal fragment (1–107) of RipAW is sufficient for triggering cell death in N. tabacum, we speculate that the R75A/R78A mutation might alter RipAW‐triggered cell death in this species. To test this idea, the RipAW (R75A/R78A) and RipAW (H62D/H64D) were expressed in N. tabacum leaves. Similar to what we observed in N. benthamiana, the H62D/H64D mutation did not alter RipAW‐triggered cell death in N. tabacum. Interestingly, the R75A/R78A mutation completely suppressed RipAW‐elicited cell death (Figure 6c,d). These results suggest that in contrast to N. benthamiana, R75 and R78 play an important role in RipAWGM‐triggered cell death in N. tabacum. These data again imply that N. tabacum and N. benthamiana use different strategies to detect RipAW and initiate ETI.

2.8. Loss of function RipAW does not change the virulence/avirulence of R. solanacearum on solanaceous plants

To determine the contribution of RipAW in GMI1000‐triggered cell death, we generated a mutant strain in which the RipAW coding sequence was completely deleted from its genome (ΔripAW), and investigated the effect of this mutation on cell death in Nicotiana spp. The ΔripAW strain was able to trigger cell death in both N. benthamiana and N. tabacum to similar levels as the wild‐type strain GMI1000 (Figure S6a). Consistent with cell death symptoms, no difference in conductivity was detected between the wild type strain and the ΔripAW strain (Figure S6b), suggesting that GMI1000‐triggered cell death was mediated by additional effectors besides RipAW. This result is consistent with previous findings demonstrating that RipB, AvrA, PopP1, and RipE1 effectors trigger cell death in N. benthamiana or N. tabacum (Jeon et al., 2020; Nakano & Mukaihara, 2019a, 2019b; Poueymiro et al., 2009; Sang et al., 2020).

Because R. solanacearum GMI1000 also causes brown rot disease on potato, we used an in vitro potato infection system to investigate whether this deletion affected the pathogen's fitness on potato plants (Wang et al., 2019). Similar to the wild‐type strain, the mutant strain caused severe wilting symptoms on potato (Figure S6c,d). In line with this, R. solanacearum wild‐type strain and ΔripAW strain reached similar growth levels in potato stems at 4 dpi, regardless of the inoculation method used (with or without wounding the roots) (Figure S6c,d). These results indicate that RipAW deficiency does not seem to affect the proliferation of R. solanacearum GMI1000 in potato plants.

3. DISCUSSION

Effectors are the most important virulence determinants of gram‐negative bacterial pathogens infecting plants. Occasionally, effectors betray the pathogen and activate ETI, resulting in failure of pathogen invasion. Except for a few examples, it remains largely unknown how different effectors together with different host targets induce ETI and cell death. Here, we characterized how RipAWGM from the bacterial wilt pathogen R. solanacearum triggered cell death in Nicotiana species (N. benthamiana and N. tabacum) in different ways. We found that the recognition of RipAW by N. benthamiana up‐regulates defence‐related gene NbPR1 expression and activates cell death and plant immunity. Thus, the cell death triggered by RipAW is dependent on NbSGT1. We also showed that RipAWRS elicits very strong cell death in N. benthamiana and N. tabacum (Figure 4). However, Nakano and colleagues reported that RipAWRS induced leaf chlorosis, but not cell death in N. benthamiana (Nakano et al., 2017). This discrepancy could be attributed to the different experimental conditions used for plant growth (23°C and 70% humidity in our assay, 25°C and 50% humidity in Nakano's assay) and/or the Agrobacterium inoculum used for leaf infiltration (OD600 = 1 in our assay and OD600 = 0.5 in Nakano's assay).

The RipAWGM protein appears as two isoforms when expressed in N. benthamiana (Figures 3e and 6e). This could suggest that RipAWGM may undergo posttranslational modification(s) on its entry to the host cells. However, these two isoforms do not always have similar intensities (Figures S2 and 6e). Because the RipAW‐FLAG size (471 amino acids) overlaps with that of the large subunit of RuBisCO, the observed two RipAW‐FLAG bands might be artificial because the RuBisCO large subunit split one band into two, a phenomenon known as a “hamburger band”. An alternative explanation is that RipAW undergoes proteolytic cleavage in planta. More experiments (such as immunoprecipitation assays) need to be performed to verify the nature of the two bands observed.

Deletion of RipAW did not have any noticeable effect on GMI1000‐triggered HR on Nicotiana species (Figure S6a,b). This indicates that besides RipAW, other effectors also contribute to the cell death phenotype observed on Nicotiana species on infection. Consistent with this, several effectors, such as AvrA, PopP1, RipAB, RipB, RipE1, and RipI, have been sequentially shown to trigger cell death in Nicotiana species (Landry et al., 2020; Poueymiro et al., 2014). Interestingly, some R. solanacearum effectors suppress cell death triggered by other effectors in Nicotiana. For example, RipAY and RipAC block RipE1‐induced Nicotiana leaf cell death by degrading glutathione and inhibiting SGT1‐mediated MAPK activation (Sang et al., 2020; Yu et al., 2020). RipAK inhibits R. solanacearum‐triggered cell death on N. tabacum (Sun et al., 2017). It will be interesting to know the inhibition spectrum of these effectors on the known R. solanacearum effector‐triggered cell death. In potato, RipAW deficiency did not decrease GMI1000 pathogenicity (Figure S6c,d), similar to what has been previously shown in pepper and tomato (Nakano et al., 2017). Notably, overexpressing RipAW in Arabidopsis showed enhanced plant sensitivity to P. syringae DC3000, suggesting that RipAW inhibits plant immunity (Nakano et al., 2017). This implies that RipAW has functional redundancy with some of the 70 R. solanacearum effectors (Sabbagh et al., 2019). In agreement with this, lack of individual effectors in P. syringae does not change its virulence in terms of bacterial growth in leaves and bacterial symptoms (Kvitko et al., 2009). In addition, restoration of most individual effectors to a DC3000 mutant strain lacking 28 effectors does not significantly enhance its virulence on plants (Cunnac et al., 2011). Similarly, disruption of single R. solanacearum effectors showed a very marginal effect on R. solanacearum virulence on plants (Cunnac et al., 2004; Sole et al., 2012). In addition, successful natural infection by R. solanacearum is a very complex process that has been divided into four steps: root colonization, vertical movements from roots to shoots, circular vascular bundle invasion, and radial apoplast spread in the cortex (Planas‐Marques et al., 2020). Because wilting symptoms and large bacterial multiplication occur at late stages of R. solanacearum infection, we cannot exclude the possibility that RipAW plays a role in root colonization or bacterial movements inside the sensitive plant.

In this study, we proved the E3 ligase activity of RipAWGM is essential, but not sufficient for triggering cell death on N. benthamiana (Figure 2). On the contrary, the first 107 amino acids of RipAW are sufficient for cell death induction in N. tabacum, but not in N. benthamiana (Figure 2). Furthermore, mutation R72A/R78A blocks RipAW‐triggered cell death in N. tabacum, but not in N. benthamiana (Figure 6). Together, these results indicate that N. tabacum and N. benthamiana employ different surveillance systems for sensing the effector RipAW and activating cell death and ETI. It will be very interesting to further explore how N. benthamiana and N. tabacum differentially perceive RipAW and activate ETI. Furthermore, although RipAWPo and RipAWCF escaped surveillance by the N. benthamiana immune system, they were still detected by N. tabacum. This suggests that the recognition spectrum of N. tabacum on RipAW might be broader than that of N. benthamiana. In the future, identifying the NLR proteins responsible for detecting RipAWs will help in deciphering the co‐evolution strategies of NLR proteins and their cognate effectors during the long‐lasting battle between host and pathogens. This will provide new insights into the molecular mechanism underlying recognition of allelic effectors by NLRs in different plant species during plant–microbe co‐evolution and valuable information for breeding highly resistant crops based on NLR‐protein mediated plant immunity.

4. EXPERIMENTAL PROCEDURES

4.1. Plant materials and microbial strains

Nicotiana spp. plants were sown in soil and grown in controlled chambers under 23°C and short‐day conditions with 70% humidity. Potato explants of cv. Desirée were grown in liquid Murashige & Skoog medium with 20% sucrose (MS2) at 22–23°C, 70% humidity, and long‐day conditions.

R. solanacearum strains GMI1000, CIP301, CFBP2957, UW551, IPO1905, NCPPB3987, and UY031 are described in Lu et al. (2018). Wild‐type R. solanacearum JY1 strain was isolated from wilting tobacco plants (Yunyan 87) at Jiangyong county, Hunan province, China. The wild‐type JY1 strain was transformed with a pHM1 plasmid which confers the ability of JY1 to grow on the spectinomycin‐amended medium. All strains used in this study were grown at 28°C on solid or liquid rich B medium (1% Bacto peptone, 0.1% yeast extract, and 0.1% casamino acids; Becton Dickinson) with the appropriate antibiotics.

4.2. Plasmid constructions

All primers used in this study are listed in Table S1. Genomic DNA of R. solanacearum GMI1000 was used as the template for PCR amplification of RipAW and its derivatives, which were cloned into pCambia 6604‐GFP (BamHI and SalI/XhoI) (Bai et al., 2016) or pCambia1300‐FLAG (KpnI/SacI and BstBI) with a NovoRes plus one‐step cloning kit (Novoprotein Scientific Inc.). The RipAW orthologs from strains RS1000, CFBP2957, PSI07, and Po82 were synthesized and cloned into pUC57 by the Gene Create company in Wuhan, China. These constructs were then directly digested with KpnI and BstBI and subcloned into pCambia 1300‐FLAG with KpnI and BstBI. All plasmids made in this study are listed in Table S2.

4.3. Construction of ΔRipAW strain

A 539 bp fragment at the upstream of RipAW start codon and a 682 bp fragment downstream of the RipAW stop codon were amplified from genomic DNA of R. solanacearum GMI1000 by chimeric PCR and were cloned into pURF80 vector. The resulting construct was introduced into R. solanacearum GMI1000 by natural transformation (Valls et al., 2006). The colonies grown on B medium containing 50 µg/ml kanamycin were selected and cultured in liquid B medium without antibiotics overnight. Cell cultures were spread on solid B medium containing 5% sucrose. Positive ΔRipAW candidates were confirmed by PCR.

4.4. Ubiquitination assay

R. solanacearum GMI1000 RipAW was cloned into pGEX6p‐1 vector with BamHI/XhoI. PCR‐based site‐directed mutagenesis was used to mutate the cysteine 177 of RipAW into an alanine or a serine. The obtained pGEX‐RipAW, pGEX‐RipAW (C177A) and pGEX‐RipAW (C177S) were transformed into E. coli BL21. The GST‐tagged RipAW, RipAW (C177A), and RipAW (C177S) were purified from the IPTG‐induced cell cultures by glutathione resin beads (Sigma) and detected by western blot with anti‐GST antibody (Sigma).

Two micrograms of purified GST‐tagged RipAW and its derivatives were incubated with 0.5 μg human E1, 2 µg UbcH5B E2, and 2 µg HA‐ubiquitin (Boston Biochem) in 40 µl of reaction buffer (25 mM Tris.HCl pH 7.5, 50 mM NaCl, 5 mM ATP, 10 mM MgCl2, 0.1 mM dithiothreitol) for 2 h at 37°C. The reaction was stopped by addition of 5× SDS‐loading buffer. The reaction mixtures were separated by SDS‐PAGE and detected with anti‐GST antibody and anti‐HA antibody (Sigma).

4.5. Agrobacterium‐mediated transient expression assay

Gene transient expression in Nicotiana species was performed as previously reported (Grosse‐Holz et al., 2018). Agrobacterium tumefaciens GV3101 carrying RipAW and its derivatives were cultured in liquid LB medium at 28°C overnight, then collected by centrifugation and suspended with the inoculation buffer (10 mM MES pH 5.0, 10 mM MgCl2, 400 nM acetosyringone). Five‐week‐old N. benthamiana and N. tabacum were infiltrated with Agrobacterium at an optical density of 1 (OD600 = 1) and covered with a transparent plastic membrane for 2 days and kept in a chamber under 23°C and long‐day conditions with 70% humidity for gene expression.

4.6. Cell death and ion leakage assay

To visualize cell death triggered by RipAW and its mutants on leaves, photographs of the leaves of N. benthamiana and N. tabacum inoculated with agrobacteria were taken with a digital camera at 4–5 dpi. For electrolyte leakage measurements, 15 leaf discs per sample were harvested, put into three tubes containing 5 ml of double‐deionized water (five leaf discs/tuber), and subjected to vacuum for 20 min (Szczesny et al., 2010). Then all samples were left at room temperature for 2 h and the ion leakage caused by RipAW and its derivatives was measured with a conductimeter. To determine total conductivity of the entire sample, conductivity was measured again after boiling the solution for 20 min (Szczesny et al., 2010). Quantification of cell death triggered by RipAW and its mutants was displayed as the percentage of Conductivityunboiling/Conductivityboiling.

4.7. Virus‐induced gene silencing assay

An equal volume mixture of Agrobacterium (OD600 = 1) containing TRV2::NbSGT1, TRV2::GFP, and TRV2::NbPDS with Agrobacterium (OD600 = 1) carrying TRV1 was infiltrated into the first two true leaves of 2‐week‐old N. benthamiana, which were then covered in plastic film for 2 days to keep high humidity. After that, the inoculated plants continued growing under 23°C and short‐day conditions with 70% humidity for another 3 weeks. When plants inoculated with TRV2::NbPDS turned completely white (Senthil‐Kumar et al., 2007), TRV2::GFP and TRV2::NbSGT1 gene‐silenced plants were agroinfiltrated with RipAW and its derivatives. After 3–4 dpi cell death phenotypes were investigated.

4.8. R. solanacearum infection assay

R. solanacearum strains were grown at 28°C in liquid rich B medium overnight. Bacteria were collected by centrifugation and washed with sterile double‐deionized water and adjusted to a final OD600 = 0.01. Five‐ to 6‐week‐old N. benthamiana plants were directly infiltrated with the bacterial solution. The inoculated N. benthamiana plants were put back into the infection chamber at 26°C, 75% humidity, and a 16 h light and 8 h dark photoperiod. Cell death symptoms were recorded at 3 dpi with a digital camera and quantified with electrolyte leakage (described above).

For potato explants infection we followed the protocol as described in Wang et al. (2019). Briefly, the roots of 2‐ to 3‐week‐old potato plants grown in liquid MS2 medium were cut with scissors and plants were subsequently transferred into glass jars containing 40 ml of a solution of R. solanacearum (OD600 = 0.01) and kept in the infection chamber under 26°C, 75% humidity, and 16 h light and 8 h dark photoperiod conditions. Photographs were taken with a digital camera and bacterial loads in the aerial part of the infected plants were measured when plants started wilting (Wang et al., 2019).

To measure R. solanacearum growth in N. benthamiana, we followed a R. solanacearum infection protocol (Sang et al., 2020) with minor modifications. Briefly, leaves were first infiltrated with agrobacteria containing RipAW (OD600 = 0.01), which is resistant to kanamycin. Around 20 h after agroinfiltration, a 105 cfu/ml solution of R. solanacearum JY1 with resistance to spectinomycin was inoculated by infiltration into the leaves expressing RipAW. R. solanacearum JY1 was isolated from wilting tobacco plants (Yunyan 87) at Jiangyong county, Hunan province, China and is also pathogenic on N. benthamiana (unpublished data). Twelve leaf discs were collected, weighed, and homogenized in sterile double‐deionized water. Bacterial cfu were counted after plating on B medium with spectinomycin.

4.9. Gene expression assay

Total RNA from leaves of N. benthamiana at the indicated times were extracted with TRIzol reagent (Invitrogen) and the first‐strand cDNA was synthesized with an EvoM‐MLV RT kit (Accurate Biotechnology). Five microlitres of the 20‐fold diluted cDNA and primers (Table S1) was directly added into the SYBR Green Mix (Gene Star Co.). The prepared quantitative PCR (qPCR) solutions were run on a iQ7 real‐time cycler (Life Technologies). Gene expression was quantified and normalized to NbActin using the 2−ΔΔ C t method.

4.10. Statistical analysis

Statistical analyses were performed using Graphpad Prism v. 7.0 software. All statistical tests are indicated in the respective figure legends.

Supporting information

 

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ACKNOWLEDGEMENTS

We are grateful to Dr Yu Du (Northwest A&F University) for the constructs TRV1, TRV2::GFP, TRV2::NbSGT1, and TRV2::NbPDS, and to Dr Changgen Xie for pCambia6604‐GFP vector and Dr Wu Chen (College of Plant Protection, Hunan Agricultural University) for R. solanacearum JY1. We also thank the Crop Biology Innovation Platform in the Agronomy College and Public Platform in the State Key Laboratory of Crop Stress Biology for Arid Areas at Northwest A&F University, China. This work was funded by the Nature Science Basic Research Plan of Shaanxi Province (2020JM‐160), the Special Research project from Shaanxi province (F2020221001), the Program of Introducing Talents of Innovative Discipline to Universities (Project 111) from the State administration of Foreign Experts Affairs (B18042) and the External Science and Technology Cooperation Program of Ningxia Academy of Agriculture and Forestry Sciences (DW‐X‐2018012), the Start‐up Funds of Northwest A&F University (Z111021601), the Fundamental Research Fund for the Central Universities of China (Z109021706), the Key Research and Development plan of Ningxia Province (2021BEF02033), and the National Natural Science Foundation of China for C.Z. (31901573).

Niu, Y. , Fu, S. , Chen, G. , Wang, H. , Wang, Y. , Hu, J. , et al (2022) Different epitopes of Ralstonia solanacearum effector RipAW are recognized by two Nicotiana species and trigger immune responses. Molecular Plant Pathology, 23, 188–203. 10.1111/mpp.13153

Yang Niu, Shouyang Fu, Gong Chen, and Huijuan Wang contributed equally.

Contributor Information

Cuizhu Zhao, Email: zhaocuizhu2002@163.com.

Qin Chen, Email: chenpeter2289@nwsuaf.edu.cn.

Haibin Lu, Email: luhaibin011@hotmail.com.

DATA AVAILABILITY STATEMENT

All data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Data Availability Statement

All data supporting the findings of this study are available from the corresponding author upon reasonable request.

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