Vacuolar processing enzyme positively modulates plant resistance and cell death in response to Phytophthora parasitica infection

GAO Xian-xian, TANG Ya-ling, SHI Qing-yao, WEI Yu-shu, WANG Xiao-xue, SHAN Wei-xing, QIANG Xiao-yu

State Key Laboratory of Crop Stress Biology for Arid Areas/College of Agronomy, Northwest A&F University, Yangling 712100, P.R.China

Abstract Oomycete, particularly Phytophthora species, causes the most devastating crop diseases, such as potato late blight, and threatens the sustainable crop production worldwide.Our previous studies identified Resistance to Phytophthora parasitica 1 (RTP1) as a negative regulator of Arabidopsis resistance to multiple biotrophic pathogens and RTP1 lossof-function plants displayed rapid cell death and reactive oxygen species (ROS) production during early colonization of P.parasitica.In this study, we aim to decipher the mechanism of RTP1-mediated cell death, and identify a member of vaculoar processing enzymes (VPEs), γVPE, playing a role in rtp1-mediated resistance to P.parasitica and cell death occurrence.Our results showed up-regulation of the expression of γVPE as well as increased VPE/caspase 1-like protease activity in P.parasitica-infected rtp1 mutant plants.Besides, we found that the VPE/caspase 1-like protease activity was required for the cell death occurrence in Arabidopsis plants during the infection of P.parasitica as well as rtp1-mediated resistance to P.parasitica.Further pathogenicity assays on either Arabidopsis γvpe mutant plants or leaves of Nicotiana benthamiana with transient overexpression of γVPE demonstrated γVPE could positively affect plant resistance to P.parasitica.Together, our studies suggest that γVPE might function as an important regulator of plant defense and cell death occurrence in response to P.parasitica infection, and VPE/caspase 1-like protease activity is required for rtp1-mediated resistance to P.parasitica.

Keywords: cell death, caspase 1, plant resistance, Phytophthora parasitica, RTP1, susceptibility factor, vacuolar processing enzyme

1.Introduction

Programmed cell death (PCD) is an active and orderly process with genetic control in multicellular organisms, and it aims to maintain the stability of the internal environment and better adapt to the surrounding environment during development, defence response and adverse conditions (Van Hautegemet al.2015; Valandroet al.2020).The regulatory mechanism of PCD is highly conserved in animals and plants, as evident by the function of caspase-like proteases in both mammalian apoptosis and plant PCD and many studies have confirmed the function of caspase-like proteases in the plant hypersensitivity reaction against pathogen (Juliánet al.2013; Kabbageet al.2017).To avoid excessive pathogen invasion, plants have developed a typical PCD in term of hypersensitive response (HR), which causes rapid local cell death at the site of pathogen infection that prevents further ingress of biotrophic pathogens that thrive on living host tissue (Muret al.2008).Therefore, cell death is considered to be an important plant defence strategy when confronting with invading pathogens (Greenberg 1997; Pitsiliet al.2020).

Vacuolar processing enzyme (VPE) belongs to a cysteine protease family protein and exhibits caspase 1-like activity with substrate-specificity for asparagine and aspartic acid, which cleaves both caspase 1-specific substrate (AC-YVAD-MCA) and VPE-specific substrate (AC-ESEN-MCA) (Hatsugaiet al.2004).It locates in plant vacuoles and is responsible for the maturation and activation of vacuolar associated proteins including seed storage proteins, proteolytic enzymes and defensive proteins (Hara-Nishimuraet al.1991, 1993; Yamadaet al.2020).As the first identified vacuolar component as a regulator of cell death, VPE-mediated vacuolar cell death was thought to be unique in plants (Hatsugaiet al.2006).By releasing hydrolytic enzymes to the cytosol from the collapsed vacuole, VPEs function as an executioner of plant cell death and contribute to maintain a balance between plant growth and immunity (Kinoshitaet al.1999; Vorsteret al.2019).Correspondingly, VPE has been identified to play an important role in biotic stress-induced plant cell death (Hatsugaiet al.2015).For instance, VPE-mediated cell death was found to be associated with tobacco resistance against viral pathogen (Hatsugaiet al.2004).In addition, VPE is also involved in the cell death induced by a fungal toxin fumonisin B1 (FB1) (Kuroyanagiet al.2005).Further studies suggest that VPE may be involved in elicitor-triggered immunityviaregulating pathogen elicitor-induced HR and elicitor-mediated stomatal closure (Zhanget al.2010).Recent findings that VPEs (β-type and γ-type VPEs) exhibit peptide ligase activity and participate in the synthesis of cyclic peptides maintaining antibiotic activity further confirm the crucial role of VPE in plant immunity (Craik 2012; Jameset al.2018; Yamadaet al.2020).

InArabidopsis thaliana, there are four types ofVPE(i.e.,αVPE,γVPE,βVPE, andδVPE).They are divided into three subfamilies including vegetativeVPE(αVPEandγVPE), seed-typeVPE(βVPE) and a novelVPE(δVPE), which is only found in dicots (Yamadaet al.2005; Vorsteret al.2019).Among them, the expressions of vegetativeVPEswere found to be up-regulated in response to stress stimuli such as wounding and pathogen infection or during plant development and senescence (Yamadaet al.2004; Hatsugaiet al.2015).Particularly, theγVPEhas been found to play a vital role in response to abiotic and biotic stresses (Rojoet al.2004; Kuroyanagiet al.2005; Liet al.2012).

Oomycete, especiallyPhytophthoraspecies, are destructive plant pathogen which threatens the food security worldwide.One typical example is the potato late blight disease caused byP.infestans, leading to huge economic costs annually (Fryet al.2015; Zhanget al.2020).As a model oomycete pathogen,P.parasiticais a typical hemibiotrophic pathogen capable of infecting a wide range of host plants, includingN.benthamianaandA.thaliana(Menget al.2014).Using the compatible interaction betweenP.parasiticaandArabidopsis, we previously identified a novel susceptibility factorResistanceToPhytophthoraparasitica1(RTP1), a nodulin-related protein, which belongs toArabidopsisMedicagoTruncatulaNODULIN21(MtN21) family.RTP1loss-of-function plants showed more resistance toP.parasitica, accompanied with rapid local cell death, strong reactive oxygen species (ROS) production andPR1expression (Panet al.2016).Nevertheless, as an important plant defence strategy, the execution and mechanism of cell death mediated by RTP1 remain unclear.

In this study, we aim to investigate the mechanism of RTP1-mediated cell death in response toP.parasiticainfection.Our results suggest that VPE/caspase 1-like enzymatic activity is required for the cell death occurrence inArabidopsisplants during the infection ofP.parasiticaas well asrtp1-mediated resistance toP.parasitica.Moreover, our pathogenicity assays on eitherArabidopsisγvpemutant plants or leaves ofN.benthamianawith transient overexpression ofγVPEdemonstrate thatγVPEmight positively affect plant resistance toP.parasitica.Taken together, our studies expand the role of VPE in plant cell death and resistance in response toPhytophthorainfection and facilitate our understanding of the cell death mechanism mediated by the susceptibility factor RTP1.

2.Materials and methods

2.1.Plant materials and growth conditions

Arabidopsisthalianaof ecotype Columbia-0 (Col-0) plants and T-DNA insertion mutants in the Col-0 background were used.TheRTP1(GenBank accession number AT1G70260) T-DNA insertion line was obtained from the Arabidopsis Biological Resource Center (ABRC):rtp1-1(SALK_094320).Theγvpemutant was provided by Dr.Patrick Sch?fer, University of Giessen, Germany.To carry out either pathogen colonization or enzymatic activity assays in the roots ofA.thalianaCol-0,γvpemutant andrtp1mutant seedlings, seeds were stratified at 4°C for 2–3 d before germination, and plants were grown under continuous white light at (23±2)°C on 1/2 Murashige and Skoog (MS) medium (1.5% sucrose, 0.8% agar).For leaf inoculation or treatment on leaves ofA.thalianaandN.benthamiana, the plants were grown at 23°C with 14 h of light per 24 h.

2.2.RNA extraction and RT-qPCR analysis

For RT-qPCR analysis, 2-wk-old seedlings were inoculated withP.parasitica.Total RNA was extracted from root material by using TRIZOL (Invitrogen, USA) according to the manufacturer’s instructions.A total of 1 μg of total RNA was reverse transcribed using the PrimeScript RT Reagent Kit (TaKaRa, Japan).For realtime qPCR analysis, 20 ng of the first-strand cDNA reaction products was used as template in a reaction with Ultra SYBR Mixture (CWBIO, China) under the following conditions: 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 30 s.The fold changes in gene expression were normalized usingAtUBC9as the internal control.Primers used for RT-qPCR are listed in Appendix A.

2.3.VPE/caspase 1-like protease activity assay

VPE/caspase 1-like protease activity was measured using the method reported by Kuroyanagiet al.(2005).The 2-wk-oldArabidopsisroots of above transgenic lines were inoculated withP.parasiticaor mock treated, and the roots were harvested at indicated time points during the early biotrophic colonization phase.The roots of above transgenic lines were preincubated in 100 mmol L–1sodium acetate, pH 5.5, containing 100 mmol L–1NaCl, 1 mmol L–1EDTA and 1 mmol L–1phenylmethylsulfonyl fluoride.The homogenate was centrifuged at 15 000×g for 10 min at 4°C, and each fluorogenic substrate of ACYVAD-MCA and AC-ESEN-MCA (substrate of caspase 1 and VPE, respectively; Peptide Institute, Japan) was added to the supernatant for measuring the VPE/caspase 1 activities.The fluorescence intensities were measured at 460 nm with a fluorescence microplate reader (TECAN infinite 200, Switzerland).

2.4.Agrobacterium-mediated transient expression in leaves of N.benthamiana

AgrobacteriumtumefaciensGV3101 strain harboring the recombinant constructs were grown for 1 d at 28°C in LB medium in the presence of corresponding antibiotics.Afterwards, the bacterial cells were harvested by centrifugation, and resuspended in MES buffer to OD600=0.4 and incubated for 1 h before infiltration.The 4-wk-oldN.benthamianaleaves were infiltrated using 1 mL syringe without needle from the abaxial side.And 2 d after infiltration, leaves were collected to inoculate withP.parasitica.Lesion area was evaluated at 36 h post infiltration (hpi).

2.5.Trypan blue staining and microscopic characterization

In order to determine the progress of infection or cell death, inoculated leaves were stained with trypan blue solution (10 g phenol, 10 mL glycerol, 10 mL lactic acid, 10 mL water, and 10 mg trypan blue) by boiling for 2 min.After cooling to room temperature, the samples were destained with chloral hydrate solution.The samples were rinsed with water and visualized under an Olympus Microscope (Japan).

2.6.Determination of cell death rate

Cell death rate was determined using the method reported by Qianget al.(2012).The 2-wk-oldArabidopsisroots infected withP.parasiticawere stained by FDA.Roots were washed with PBS buffer before transferred to the PBS buffer containing FDA at a final concentration of 5–10 μmol L–1.After incubation for 15 min, roots were washed twice by the same buffer, fluorescence intensities were measured at 535 nm after excitation at 485 nm by a fluorescence multimode reader (Victor Nivo, Finland).

2.7.Plasmid construction

The full-length coding sequences ofαVPEandγVPEwere amplified using PCR from the Col-0 cDNA library.In order to create the expression cassette, theαVPEandγVPEwas fused at the C terminus with the 4× MYC sequence and inserted into theXhol andHindIII sites of pKannibal (Wesleyet al.2001), then inserted into the binary vector pART27 (Gleave 1992) at theNotI site.The open reading frame ofαVPEandγVPEgene was introduced into the binary vector pART27 under the 35S-promoter respectively to generateαVPEandγVPEoverexpression constructs.

2.8.Phytophthora parasitica infection assay

TheP.parasiticastrain Pp016 was used for plant infection.TheP.parasiticaculture and zoospore preparation were performed as in previous reports (Wanget al.2011).For detached leaves inoculation, the fully expanded apical leaves from approximately 4-wk-oldArabidopsisplants were dip-inoculated withP.parasiticazoospores as previously described (Panet al.2016).For root inoculation, roots of 2-wk-old seedlings were dipinoculated withP.parasiticazoospores (Panet al.2016).Specific primers forP.parasiticaUBC(PpUBC) were used to monitor the level of pathogen colonization andAtUBC9was used to indicate plant biomass.Results were presented as a proportion between pathogen and plant genomic DNA to reveal the extent of infection.

2.9.Dual-luciferase reporter assay

The sequence encoding the activated forms of bZIP60 and bZIP28 were cloned into the pGreenII 62-SK effector vector at theBamHI andPstI sites, and the promoter ofγVPEwas cloned into the pGreenII 0800-LUC reporter vector at theHindIII andBamHI sites.The recombinant plasmids and negative control vectors were introduced intoA.tumefaciensGV3101 (pSoup-p19).The effector and reporter vectors were co-transformed intoN.benthamianaleaves, as previously described (Yaoet al.2020).LUC (Firefly luciferase) and REN (Renilla luciferase) activities were measured at 2 d after infiltration, using the Dual Luciferase Reporter Assay Kit (Promega, USA).

3.Results

3.1.Induction of αVPE and γVPE is accompanied with increased cell death occurrence in RTP1 loss-of-function plants upon early colonization of P.parasitica

To investigate whetherRTP1is involved in regulating the dynamic expression ofVPEsduring the early colonization ofP.parasitica, 2-wk-old WT Col-0 andrtp1mutant seedlings were inoculated withP.parasiticazoospores and the transcript levels ofVPEs(αVPE,βVPEandγVPE) in both WT Col-0 andrtp1mutant roots were analyzed at 3, 6, 12 and 24 hpi, respectively, by RT-qPCR.The results showed that bothαVPEandγVPEwas not significantly induced until 12 and 24 hpi, at which the expression levels of bothαVPEandγVPEwere significantly elevated inrtp1mutants, compared with those in WT Col-0 (Fig.1-A and B).On the contrary, the expression ofβVPEwas not obviously induced (Appendix B).These results indicate thatRTP1might negatively regulate the expression ofγVPEandαVPEduring the early colonization ofP.parasitica.

To relate the induction of expression of theseVPEgenes to colonization-associated cell death, we performed a fluorescein diacetate (FDA)-based cell viability assay onP.parasitica-infected roots of WT Col-0 andrtp1mutant plants at 3, 6, 12 and 24 hpi, respectively.As esterases cleave off fluorescein in living cells, the degree of cleavage can be quantified spectrometrically.Through analysis on the cell death rate in infected root tissues at indicated time points, we found that the occurrence of cell death was significantly enhanced inrtp1mutant plants compared with WT Col-0 during the early colonization ofP.parasitcia(Fig.1-C).These results indicate a correlation of induction ofγVPEandαVPEwith the occurrence of enhanced cell death inRTP1loss-offunction plants upon early infection byP.parasitica, suggesting the role ofγVPEandαVPEinRTP1-mediated cell death in response to attempted infection byP.parasitica.

Fig.1 Expression patterns of VPE genes and cell death occurrence in Arabidopsis thaliana roots upon early colonization of Phytophthora parasitica.A and B, expressions of αVPE and γVPE in 2-wk-old A.thaliana WT Col-0 and rtp1 mutant roots were evaluated by RT-qPCR at indicated time points.Data represent fold changes of genes and display the ratio of candidate gene expression to plant housekeeping gene AtUBIQUITIN9 using the 2–ΔΔCt method in colonized plants relative to mock-treated plants.Fold changes >1 indicate induction of genes.Error bars represent SD (n=200) .Statistical significance was assessed by Student’s t-test (＊＊, P<0.01).Similar results were observed in at least 3 independent experiments.C, FDA-based cell viability assay indicative of cell death in P.parasitica infected roots.The values are given as relative fluorescence units relative to mock-treated roots.Data are mean±SD of 8 independent measurements per treatment of 4 independent biological experiments.

3.2.RTP1 loss-of-function might contribute to enhancing plant VPE/caspase 1-like activities

As both VPE- and caspase 1-like protease activities are required for vacuole-mediated plant cell death execution (Hatsugaiet al.2004; Kuroyanagiet al.2005), we were prompted to examine the occurrence of these protease activities during early colonization ofP.parasiticaand explore the impact of RTP1 on the protease activities.To this end, we measured VPE- and caspase 1-like activities in roots of WT Col-0 andrtp1mutants during biotrophicassociated colonization (6 and 24 hpi).Upon addition of either VPE-specific substrate Ac-ESEN-MCA or caspase 1-specific substrate Ac-YVAD-MCA to root extracts, VPE-mediated cleavage of MCA was spectrometrically determined.In comparison to root extracts of WT Col-0 with control treatment, we found significantly enhanced VPE/caspase 1 activities in root extracts ofP.parasiticainfected WT Col-0 (Fig.2-A and B), indicating that the enzymatic activities were activated byP.prasiticainfection.Notably, we found generally higher levels of enzymatic activities in root extract ofrtp1mutants than those in WT Col-0, andrtp1mutant showed significantly higher level of VPE/caspase 1 enzymatic activities duringP.parasiticainfection, particularly at 24 hpi (Fig.2-A and B).Taken together, these results implied thatRTP1lossof-function contributes to enhanced VPE/caspase 1-like activities during theP.parasiticainfection.

Fig.2 Quantification of VPE/caspase 1 enzymatic activities in Phytophthora parasitica-infected Arabidopsis thaliana roots.VPE (A) and caspase 1 (B) activities were examined in A.thaliana WT Col-0 and rtp1 mutant roots infected by P.parasitica at indicated time points.VPE-specific substrate Ac-ESEN-MCA or caspase 1-specific substrate Ac-YVADMCA was added to the root extracts for spectrophotometric determination of VPE and caspase 1 activity, respectively.Data are mean±SD of 8 independent measurements per treatment of 3 independent biological experiments.Statistical significance was assessed by Student’s t-test (＊＊, P<0.01).

3.3.VPE/caspase 1-like activities play a pivotal role in response to P.parasitica infection

To investigate whether VPE/caspase 1-like activity is of importance for the occurrence of plant cell death and resistance againstP.parasitica, we infiltrated a caspase 1 inhibitor (Ac-YVAD-CMK) into leaves of 4-wk-old WT Col-0 prior to inoculation withP.parasitica.To detect the occurrence of cell death, leaves were stained with trypan blue at 12 hpi and examined by microscopy.The results showed that invasive hypha extended in leaves infiltrated with 0.1 mmol L–1DMSO control solution at 12 hpi (Fig.3-A), whereas less cell death occurred in leaves infiltrated with 0.1 mmol L–1concentration of caspase 1 inhibitor (Fig.3-B).These results indicate that VPE/caspase 1-like activity plays a pivotal role in plant cell death during the early colonization ofP.parasitica.

We next examined the effect of VPE/caspase 1-like activities on the colonization ofP.parasiticaonArabidopsisplants.For this, we first infiltrated the caspase 1 inhibitor (Ac-YVAD-CMK) and control solution into detached leaves of 4-wk-old WT Col-0 prior to inoculation withP.parasiticazoospores.At 3 dpi, we found that leaves infiltrated with caspase 1 inhibitor displayed severer water-soaked lesions compared with control leaves (Fig.3-C).Further qPCR assay for pathogen biomass consistently indicated that the infiltration with caspase 1 inhibitor conferred plant more susceptibility than control (Fig.3-D).These results imply that VPE/caspase 1-like activity is of importance to plant resistance againstP.parasitica.

Fig.3 VPE/caspase 1-like activity affects plant cell death and resistance to Phytophthora parasitica.A and B, either the mock solution containing 0.1 mmol L–1 DMSO (A, mock) or 0.1 mmol L–1 caspase 1 inhibitor Ac-YVAD-CMK (B, CMK) was infiltrated into the leaves of 4-wk-old Col-0 plants, prior to the inoculation with P.parasitica zoospores.The infected leaves were stained with trypan blue to indicate cell death symptoms at 12 h post infiltration (hpi).Scale bars=100 μm.C, the leaves were photographed under normal light (upper panel) or UV illumination (lower panel) at 3 dpi to indicate the disease symptoms.D, qPCR with primers specific for the Arabidopsis thaliana UBC9 gene (AtUBC9) and the P.parasitica UBC gene (PpUBC) were used to determine P.parasitica biomass in infected leaves of Col-0 at 3 dpi.The relative P.parasitica biomass was calculated by PpUBC/AtUBC and normalized using the value of Col-0 infiltrated with mock solution.Data are mean±SD (n=20), and asterisks indicate statistical significance based on Student’s t-test (＊＊, P<0.01).

3.4.γVPE plays a positive regulatory role in plant resistance against P.parasitica

To further identify the immune function of VPE in response to colonization ofP.parasitica, we first infiltrated either construct p35S::γVPEor p35S::αVPEand the empty vector as a control into leaves ofN.benthamianaprior to inoculation withP.parasitica.At 36 hpi, we found significantly smaller infection lesions and less amount of pathogen biomass in the leaf region infiltrated with p35S::γVPErelative to control (Fig.4-A–C).By contrast, the disease symptom seemed unaltered when overexpressedαVPE(Appendix C).These results indicate thatγVPEmight play a key role in plant resistance toP.parasitica.

To complement this, we next dip-inoculated roots of WT Col-0 andγvpemutant plants withP.parasiticazoopores.The root colonization byP.parasiticawas quantified at 12 hpi (biotrophic phase) and 48 hpi (necrotrophic phase), respectively, using qRT-PCR.Theγvpemutants showed more susceptibility, with increased amount ofP.parasiticabiomass relative to WT Col-0 (Fig.4-D).Taken together, these results suggest thatγVPEmay function as a positive regulator in plant resistance againstP.parasitica.

Fig.4 AtγVPE positively affects plant resistance against Phytophthora parasitica.A, Nicotiana benthamiana leaves were infiltrated with Agrobacterium tumefaciens cells containing vectors carrying the γVPE gene or empty vector (EV), prior to the inoculation with P.parasitica zoospores.Image was taken with UV illumination at 36 h post infiltration (hpi).B, the average lesion areas at 36 hpi.Data are mean±SD of 20 infections from at least 10 leaves.C, qPCR with primers specific for the N.benthamiana β-actin and the P.parasitica UBC gene (PpUBC) were used to determine P.parasitica biomass in infected leaves of N.benthamiana at 36 hpi.The relative P.parasitica biomass was calculated by PpUBC/β-actin and normalized using the value of EV control.Data are mean±SD (n=10).D, qPCR with primers specific for the A.thaliana UBC9 gene (AtUBC9) and the P.parasitica UBC gene (PpUBC) was used to determine P.parasitica biomass in roots of A.thaliana Col-0 and γvpe mutant at 12 and 48 hpi.The relative P.parasitica biomass was calculated by PpUBC/AtUBC and normalized using the value of Col-0.Data are mean±SD (n=20), and asterisks indicate statistical significance based on Student’s t-test (＊＊, P<0.01).Similar results were obtained for at least 3 individual experiments.

3.5.The rtp1-mediated resistance to P.parasitica is in a VPE/caspase 1-like dependent manner

To examine the role of VPE/caspase 1-like activity inrtp1-mediated resistance toP.parasitica, we infiltrated the caspase 1 inhibitor (Ac-YVAD-CMK) and control solution, respectively, into detached leaves of 4-wk-oldrtp1mutant and WT Col-0 plants prior to inoculation withP.parasitica.We next examined the colonization ofP.parasitica, and found that leaves ofrtp1mutants infiltrated with control solution exhibited significantly less infection lesion than WT Col-0, whereas this resistance phenotype seemed manipulated in leaves ofrtp1mutants infiltrated with caspase 1 inhibitor, as indicated by severer watersoaked lesions thanrtp1mutants with control treatment (Fig.5-A).Further qPCR assay for pathogen biomass consistently showed that the infiltration with caspase 1 inhibitor strongly attenuatedrtp1-mediated resistance toP.parasitica, which led to much moreP.parasiticacolonization onrtp1plants relative to WT Col-0 plants (Fig.5-B).Taken together, these results imply thatrtp1-mediated resistance againstP.parasiticais in a VPE/caspase 1-like dependent manner.

Fig.5 VPE/caspase 1-like activity plays a role in rtp1-mediated resistance to Phytophthora parasitica.A, either the mock solution containing 0.1 mmol L–1 DMSO (mock) or 0.1 mmol L–1 caspase 1 inhibitor Ac-YVAD-CMK (CMK) was infiltrated into the leaves of 4-wk-old Arabidopsis thaliana Col-0 and rtp1 mutant plants, prior to the inoculation with P.parasitica zoospores.The infected leaves were photographed (upper pannel) at 3 dpi and trypan blue staining (lower panel) was performed to indicate the disease symptoms.Similar results were obtained from at least 3 individual experiments.B, qPCR of P.parasitica biomass in inoculated leaves of A.thaliana Col-0 and rtp1 mutant plants infiltrated with either mock solution or caspase-1 inhibitor by qPCR.Data are mean±SD (n=20), and asterisks indicate statistical significance based on Student’s t-test (＊＊, P<0.01).Similar results were obtained from at least 3 individual experiments.

4.Discussion

Plants have evolved a complex immune system to detect potential invaders and respond appropriately (Pitsiliet al.2020).Studies on the interaction between plant and pathogens have largely focused on the identification of plant resistance genes, which could be ultimately applied in crop resistance breeding.However, the loss ofR-gene-mediated resistance would inevitably happen due to the virulence variation of pathogens (Gorshkov and Tsers 2021).It’s worth noting that the modification of susceptibility (S)-gene turned out to be an effective strategy to confer durable disease resistance in crop plants (Engelhardtet al.2018).For instance,MLOwas reported to confer barely susceptibility to powdery mildew disease, whereasMLOloss-of-function could enhance plant durable resistance to broad-spectrum pathogens (Vogel and Somerville 2000; Kusch and Panstruga 2017).Therefore,mlo-based resistance has been applied in crop resistance breeding.Interestingly, further analysis suggested that cell death might be associated withmlobased resistance (Kusch and Panstruga 2017), which indicated the important role of cell death in susceptibility factor-mediated plant immunity.RTP1was identified to negatively affect plant resistance to multiple biotrophic pathogens by regulating plant cell death (Panet al.2016).However, the mechanism ofRTP1-mediated cell death duringP.parasiticainfection remained unknown.

In this study, we found that the increase of cell death occurrence was associated with the up-regulation ofαVPEandγVPEinRTP1loss-of-function plants during the biotrophic infection byP.parasitica(Fig.1-A and B).These results implied thatRTP1might negatively regulate the expression ofαVPEandγVPEand that VPE might play a role inRTP1-mediated cell death during the pathogen’s biotrophic infection phase.Notably, we realized very slight induction of bothαVPEandγVPE, but much higher cell death occurrence inrtp1plants at very early infection phase (i.e., 3 and 6 hpi) (Fig.1).These results prompted us to speculate that besides VPEs, there might be other co-regulators participating in RTP1-mediated cell death at very early infection stage.As one of plant proteases with casapase-1 activity, VPE was characterized to cleave the protease substrates ESEN and YVAD inN.benthamianaandArabidopsis(Hastugaiet al.2004).InA.thaliana, bothαVPEandγVPEwere up-regulated in senescing tissues and by various stress stimuli, which indicated their role in plant PCD and in responding to stresses (Kinoshitaet al.1999; Rojoet al.2004; Juliánet al.2013).Notably, further studies showed thatγVPEmight regulate vacuole-mediated cell dismantling during cell death progression in plant–pathogen interaction (Rojoet al.2004).Besides, γVPE was also confirmed to play an important role in the endophyticP.indicacolonization associated cell death (Qianget al.2012).Nevertheless, the expression ofβVPEseemed down-regulated inP.parasitica-infectedrtp1plants (Appendix B), which indicated thatβVPEmight maintain antagonistic function inRTP1-mediated cell death.Similarly, different from the activation of VPE/caspase 1-like-mediated cell death by the root endophyteP.indica,βVPEwas speculated to antagonize cell death signaling since it showed a tendency to higher fungal colonization (Qianget al.2012).

Indeed, our results showed that treatment of the specific protease inhibitor contributed to reducing the cell death occurrence and conferred more susceptibility inP.parasitica-infected Col-0 plants (Fig.3-A–D), which pointed to the notion that VPE-mediated cell death might be co-related with plant resistance toP.parasitica.Further pathogenicity assays showed enhanced susceptibility inγVPEloss-of-function plants (Fig.4-D), but more resistance when overexpressedγVPEin leaves ofN.benthamiana(Fig.4-A–C).These results supported thatγVPEmight positively regulate plant resistance toP.parasitica.Consistently, previous studies supported thatγVPEmight function in responding to biotic stress.For instance, γVPE contributed to attenuateArabidopsisresistance to biotrophic pathogenHyaloperonosporaarabidopsidis(Hpa) (Misas-Villamilet al.2012).Besides, knockout ofγVPErenderedArabidopsismore susceptible to multiple bacterial and fungal pathogens (Rojoet al.2004).

As one of the plant defense strategies, PCD plays an important role in restricting the colonization of pathogens in plant cells, especially for biotrophic pathogens.For instance, silencing of the helper NLR (NLR required for cell death) proteinsNRC4reduced the hypersensitive cell death mediated by a sensor NLR R1 and abolished R1-mediated plant resistance toP.infestansinN.benthamiana(Wuet al.2017).InArabidopsis, lack of BAK1-interacting receptor-like kinase 1 (BIR1)activates cell death and enhances plant resistance toHpa(Liuet al.2016), indicating the importance of cell death in plant immunity.Interestingly, previous studies demonstrated that RTP1 affects plant resistance to biotrophic pathogens possibly by regulating cell death (Panet al.2016).In this study, we found higher VPE/caspase 1-like protease activities accompanied with increased cell death occurrence inrtp1plants during the early infection byP.parasitica(Figs.1-C and 2-A and B).Correspondingly, with the inhibition of VPE/caspase 1-like activity, more susceptibility was observed inP.parasitica-infectedrtp1plants (Fig.5-A and B).These results suggest that VPE might play an important role in RTP1-mediated cell death and immunity.As a typical hemi-biotrophic pathogen,P.parasiticamaintains an early biotrophic colonization followed by a necrotrophic colonization on host plant (Menget al.2014).It remained elusive if VPE has diversified roles in different infection stages.As RTP1 might exert negative modulating roles in the activation of the membrane-associated transcription factors bZIP28 and bZIP60, which are required forrtp1-mediated resistance toP.parasitica(Qianget al.2021), we speculated that the expression ofγVPEmight be regulated by bZIP28 and bZIP60 transcription factors (TFs).Using the dualluciferase assay, we did not detect the binding of either bZIP28 or bZIP60 to the promoter ofγVPE(Appendix D).These results implied that the expression ofγVPEmight not be directly regulated by the bZIP28 or bZIP60 TF.It would be intriguing to explore in future as to the transcription regulation ofγVPEand how RTP1 affects the activation of the positive regulator of plant cell death and resistance in response to pathogen infection.

5.Conclusion

In this study, we propose thatγVPEfunctions as an important regulator of plant defense and cell death occurrence in response toP.parasiticainfection, and VPE/caspase 1-like protease activity is required forrtp1-mediated resistance toP.parasitica(Fig.6).Our data showed that the colonization ofP.parasiticainduced the expression ofγVPEaccompanied with increased cell death occurrence and VPE/caspase 1-like protease activity inRTP1loss-of-function plants.Besides, VPE/caspase 1-like protease activity was required for the cell death occurrence inP.parasitica-infectedArabidopsisplants andrtp1-mediated resistance toP.parasitica.Furthermore,γVPEcould positively modulate plant resistance toP.parasitica.Taken together, the results of the present studies expanded the function of VPE in plant cell death and resistance in response toPhytophthorainfection and facilitated understanding of the cell death mechanism mediated by the susceptibility factor RTP1.

Fig.6 The colonization of Phytophthora parasitica activates the expression of γVPE and VPE/caspase 1-like activities in RTP1 loss-of-function plants.γVPE, which maintains VPE/caspase 1-like activities, might play a role in plant cell death and resistance to P.parasitica.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31872657), the National Key R&D Program of China (2017YFD0200602-2), the Chinese Universities Scientific Fund (2452020146), the China Agriculture Research System (CARS-09), and the Program of Introducing Talents of Innovative Discipline to Universities (Project 111) from the State Administration of Foreign Experts Affairs, China (B18042).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2022.08.124