PnSCR82,a small cysteine-rich secretory protein of Phytophthora nicotianae,can enhance defense responses in plants

WANG Ya-di,Ll Fei,ZHANG Xin,LlU Ting-li,LlANG Wen-xing,Ll De-long

1 College of Plant Health and Medicine,Key Laboratory of Integrated Crop Pest Management of Shandong Province,Qingdao Agricultural University,Qingdao 266109,P.R.China

2 Hunan Academy of Agricultural Sciences,Changsha 410125,P.R.China

3 Provincial key laboratory of agrobiology,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,P.R.China

Abstract A number of plant pathogenic species of Phytophthora are known to produce different classes of secretory proteins during interactions with their hosts. Although several small cysteine-rich (SCR) secretory proteins,conserved in oomycete pathogens,have been identified in Phytophthora,their specific involvement in these interactions remains unknown.In this study,an SCR effector encoded by PnSCR82 in P.nicotianae was identified and shown to have similarities to P.cactorum phytotoxic protein,PcF (Phytophthora cactorum Fragaria). Agroinfection with potato virus X vector,PnSCR82,was capable of inducing plant hypersensitive cell death in Nicotiana benthamiana and Solanum lycopersicum.Real-time PCR results indicated that transiently expressed PnSCR82 in N.benthamiana leaves activated the jasmonate,salicylic acid and ethylene signaling pathways. Transient expression of PnSCR82 enhanced plant resisitance to P.capsici.In summary,our results demonstrated that P.nicotianae PnSCR82 elicits defensive responses in N.benthamiana and may potentially play a significant role in future crop protection programs.

Keywords:small cysteine-rich secretory protein,defense responses,resistance

1.lntroduction

The genusPhytophthora,with over 150 species of extremely destructive plant pathogens,is known for its devastating effects on agricultural crops and natural ecosystems (Davison 1998;Kroonet al.2012).The species,P.infestans,which was responsible for causing potato late blight disease,was a major culprit in the Irish famine and remains one of the most devastating pathogens in worldwide potato cultivation to this day (Mizubuti and Fry 2006;Nowickiet al.2011). Another widespread species,P.nicotianae(syn.P.parasiticaDastur),is one of the most economically important plant pathogens on a broad range of hosts,including tobacco,onion,tomato,cotton,pepper,citrus,as well as a large number of ornamentals(Gaulinet al.2007). It is responsible for tobacco black shank,one of the most destructive diseases in cultivated tobacco.At present,the use of fungicides in tobacco protection still effectively controls pathogens of tobacco,however,pathogens are resistant to the long term used fungicides.Phytophthoradiseases are responsible for 30–60 million dollars of losses per year in citrus orchards in Florida alone (Timmeret al.1989). Unfortunately,our lack of understanding regarding the mechanism of pathogenesis makes it difficult to combat these pathogens.

Phytopathogens produce a battery of secreted proteins that regulate plant immunity to promote infection (Birchet al.2006;Jongeet al.2011). As an adaptation against infection,plants utilize a two layer defense system to combat phytopathogens:pathogen-associated molecular patterns (PAMP) triggered immunity (PTI) and effectortriggered immunity (ETI) (Jones and Dangl 2006;Boller and Felix 2009;Dodds and Rathjen 2010;Monaghan and Zipfel 2012). PTI,as the first layer of plant defense,protects the hosts from infection by identifying conserved nonself molecules. As a countermeasure,successful phytopathogens deliver diverse effectors to thwart plant defense mechanisms. These effectors cause the secondary layer of defense,ETI,to target the apoplast or plant cytoplasm to inhibit the PTI.

Various species ofPhytophthoraare known to secrete several classes of effector proteins during plant–pathogen interactions. In the past decade,different types of elicitors fromPhytophthorapathogens have been reported,for example,GP42 ofP.sojae,Nep1-like proteins (NLP),cellulose-binding elicitor lectin(CBEL),small cysteine-rich proteins with particular cysteine pattern (SCR) and INF1 ofP.infestans(Mateoset al.1997;Séjalondelmaset al.1997;Kamoun 2006;Orsomandoet al.2011;Donget al.2012;Fenget al.2014;Zhanget al.2017). These elicitors induce defense reactions in the non-host plants,but little is known regarding their molecular function. NLPs have been isolated from many plantpathogenic fungi,bacteria and oomycetes (Mattinenet al.2004;Pembertonet al.2005;Motteramet al.2009). These can induce plant cell death and elicit strong immune responses in plants,and are frequently associated with plant perception of PAMPs. The CBEL glycoprotein fromP.nicotianaeis an elicitor that binds to crystalline cellulose,induces various defense responses in tobacco and triggers salicylic acid (SA)and jasmonate (JA) signaling pathways,as well as participating in cell wall deposition (Gaulinet al.2002). Purified PcF protein from the culture filtrate ofP.cactoruminduced leaf necrosis in its host strawberry and tomato plant,furthermore,the necrotic activity of culture filtrates was also found inP.nicotianae(Orsomandoet al.2001). SCR91,similarity to PcF,doesn’t exhibit any necrotic activity in the nonhost tobacco (Armstronget al.2003). ExpressingSCR74inN.benthamianashow necrotic symptoms,but necrotic activity is affected by the destruction of disulfide bond(Zhaoet al.2014). PcSCR82 fromP.capsiciinduce necrotic activity in its host hot pepper and tomato plant,but not inN.benthamiana. Mutation of PcSCR82 is significantly impaired the virulence ofP.capsicion tobacco (data not shown). The protein SCR96 plays an important role in pathogenicity ofP.cactorum,induces disease resistance in Solanaceae and is involved in reactive oxygen species (ROS) responses(Chenet al.2016). Thus,the SCR fromPhytophthoracan induce broad-spectrum plant disease resistance.

Here we report that secreted protein PnSCR82 has elicitor activity inN.benthamianaand tomato fromP.nicotianae. Intriguingly,PnSCR82 not only triggered plant defense responses,but also changed susceptibility to fungal and oomycete pathogens. This study will contribute to our understanding of the interactions betweenP.nicotianaeand tobacco,which may ultimately provide a novel strategy for effectively controlling tobacco black shank disease.

2.Materials and methods

2.1.Culture of plant and strains

Nicotiana benthamianaandSolanum lycopersicumAilsa Craig (AC) were grown in flowerpots (15 cm diameter) containing sterile soil (comprised of vermiculite)at 25°C with a photoperiod of 16 h light and 8 h dark.

The strain ofP.nicotianaeCP1 was provided by Tobacco Research Institute of Chinese Academy of Agricultural Sciences. The strains ofP.capsici(isolated from pepper Yiduhong) andP.nicotianaeCP1 were grown on 10% V8 juice agar for 1 week at 25°C in the dark (Gilchrist 1976).

2.2.Sequence analysis of SCR genes

FourSCRsequences ofP.cactorum(KT215392,KT215394,KT215393,and KT215395) published previously were performed against Phytophthora genome(Chenet al.2016). The orthologs of the SCR gene inP.sojae(P.sojaegenome sequence assembly database V3.0;Physo_496559,Physo_354716,and Physo_354683),P.infestans(P.infestansgenome sequence assembly database V1.1;Phyinf_13218,Phyinf_5653,Phyinf_13698,Phyinf_17023,and Phyinf_6797) andP.capsici(P.capsicigenome sequence assembly database V1.1;Phyca_96481,Phyca_96849,Phyca_130235,and Phyca_504195)were searched in the DOE Joint Genome Institute (http://genome.jgi.doe.gov/). The SCR sequences ofP.parasitica(XM_008899549,XP_008896468,XP_008916608,and XP_8905949) andP.megakarya(OWY95656,OWZ17337,OWY99957,and OWY92034) were downloaded from NCBI(http://www.ncbi.nlm.nih.gov/). Sequence alignments were analyzed using the ClustalW Program (Rédei 2008). The phylogenetic tree was constructed by the Neighbor-Joining method using Mega 4 Program (Kumaret al.2004) .

2.3.Agroinfiltration assay

For transient transformation ofN.benthamianaandS.lycopersicumAC,Agrobacterium tumefaciensstrain(GV3101) carrying pGR106-derived constructs was used as described by Wanget al.(2011). ThePnSCR82gene was amplified with the primers (PVXscr82F and PVXscr82R) and cloned into theClaI/NotI sites of pGR106,yielding the plant transient expression vector.ThePnSCR82gene was also cloned into the subcellular location vector pBin-GFP (Yuet al.2012). The sequences of primers used for construction of expression vectors are listed in Table 1. BAX and the INF1 vectors have the same backbone as thePnSCR82. A total of 12 leaves were inoculated on at least four plants. Symptom on the leaves was visually observed 4 or 6 days after infiltration.All experiments were repeated at least three times with similar results.

2.4.lnoculation assay

Leaves from 5–6 weeks oldN.benthamianawere inoculated by infiltration withA.tumefacienscarrying GFP(as a control) and PnSCR82 on the same leaves. After 24 h of agroinfiltration,P.capsiciwere was grown on 10% V8 juice agar for 1 week. Mycelial plugs of these pathogens with a diameter of 5 mm was inoculated on the nonwounded leaves ofN.benthamiana. The leaves were laid at 23–25°C under high luminosity for 2 days. Lesion sizes were measured 2 days after inoculation. Total DNA was extracted from pathogen infected regions 2 days after inoculation. The actin genes ofN.benthamianaandP.capsiciwere used as target genes to quantify the ratio of host-to-pathogen DNA. Each experiment was repeated at least three times.

2.5.Real-time PCR analysis

Total RNA were purified from tobacco leaves at different time periods afterPnSCR82expression using an RNA Extraction Kit (TaKaRa,Japan) following the manufacturer’s instructions. The first strand of cDNA was synthesized from total RNA using SuperScript III Reverse Transcriptase (Invitrogen,USA). Then,real-time PCR analysis was performed on a CFX Connect Real-Time PCR Detection System (Bio-Rad,USA) with SYBR PremixEx Taq? (TaKaRa,Japan). The program was as follows:95°C denaturation step for 30 s,followed by 40 cycles of 95°C for 5 s,60°C for 30 s and 72°C for 30 s. Finally,melting curve analysis was performed. TheN.benthamianageneEF-1αwas used as the reference and the qPCR primers are listed in Table 1. The expression was determined by the 2–ΔΔCTmethod.

Table 1 Primers used in this study

2.6.Callose and ROS detection

To visualize callose deposition,theleaves were stained with 0.01% aniline blue and subsequently imaged using an Olympus Microscopy System as described earlier(Schenk and Schikora 2014).

Diaminobenzidine staining (DAB) was utilized for detecting ROS accumulation.Nicotiana benthamianaleaves were soaked for 20 min with 0.5% DAB and then boiled for 30 min in 100% ethanol. The leaves were subsequently stained and later analyzed by lightmicroscopy.

2.7.Confocal microscopic analysis

After 2 d post-agroinfiltration (dpa),theN.benthamianaleaves were collected to either be photographed or be infiltrated with 15% NaCl solution for plasmolysis. The fluorescence was visualized by Leica TCS SP5X confocal microscopy (Leica,Wetzlar,Germany). The fluorescence photograph was taken with an exposure time of 534 ms.The experiment was repeated at least twice.

3.Results

3.1.PnSCR82 gene expression profiling of P.nicotianae

A small cysteine rich (SCR) protein,PnSCR82,with an open reading frame (ORF) encoding a secreted protein of 82 amino acids,was identified fromP.nicotianaeCP1. We discovered that homologues of PnSCR82 were found only inPhytophthoraand were not discovered in fungi. InP.cactorum,four SCR proteins have been identified including PcF-like SCR74,SCR96,SCR99 and SCR121. To determine the homologues of PnSCR82 in otherPhytophthora,SCR proteins were analysed from different species ofPhytophthora. Comparison of the different SCR proteins showed that PnSCR82 is similar to PcF-like SCR74 and shares the same subclassification in subgroups (Fig.1-A). Notably,two homologues of PcF-like proteins were identified inP.infestans;otherPhytophthorahave only one PcF-like protein.The PcF-like SCR74 gene family sequence-based alignment indicates that PnSCR82 ofP.nicotianaeand SCR91 ofP.infestansare highly similar (65%). There were 13 conserved amino acid residues in the entire family,including six conserved cysteines in PnSCR82(Fig.1-B),that were recognized for their important roles in maintaining the structure of its helix-loop-helix core domain (Nicastroet al.2009).

Fig.1 Phylogenetic analysis and protein sequence alignment of Phytophthora nicotianae small cysteine-rich (SCR) and its homologues in different Phytophthora species. A,phylogeny of PnSCR82 and SCR proteins from different Phytophthora species. The phylogenetic tree was generated by the MEGA 4.0 Program,using the Neighbour-Joining (NJ) method and 1 000 bootstrap replicates. B,alignment of Phytophthora PcF putative proteins. The red boxes indicate the six conserved cysteine sites for visualization.

In order to gain insights into the PnSCR82 function inP.nicotianae,the transcript abundance of PnSCR82 in diversePhytophthoradevelopmental stages was initially examined. PnSCR82 was found to be up-regulated during the germinating cyst stage and early infection stage. As shown in Fig.2,the results indicated thatPnSCR82was also up-regulated during plant–Phytophthorainteractions.

Fig.2 Expression profiles of Phytophthora nicotianae PnSCR82 genes. The expression of PnSCR82 was profiled using real-time PCR,with the synthesis of cDNA from each sample including the mycelia (MY),cyst (CY),germinating cyst (GC) and infectious stages (IF) (3,6,12 and 24 h post-inoculation (hpi) of Nicotiana benthamiana). Data are mean±SD (n=3).＊,statistical significance at P<0.05.

3.2.PnSCR82 protein induces necrosis in Solanaceous plants

To further characterize the biological function of PnSCR82 in plants,the sequence of PnSCR82 was cloned into potato virus X (PVX),and then used for transient expression inSolanaceousleaves with the infiltration ofA.tumefacienscells. The development of necrotic spots was monitored beginning 4 days post-treatment. We discovered that agroinfection with PnSCR82 showed symptoms of necrosis near the inoculation sites that were similar to symptoms of the positive control INF1 and BAX inN.benthamiana. The lack of obvious symptoms on transient expression of GFP served as a negative control. Similar experiments were conducted on 4 to 5 weeks oldS.lycopersicum,with the results showing that the transient expression of PnSCR82 in tomatoes resulted in necrosis 6 days post-treatment. Symptoms of necrosis were not found when GFP was transiently expressed in tomato leaves (Fig.3).

Fig.3 Transient assays of Phytophthora nicotianae PnSCR82 in Solanaceae plants. Tobacco and tomato leaves were infiltrated with Agrobacterium tumefaciens carrying PnSCR82.Phytophthora infestans INF1 elicitin causes the hypersensitive response (HR) in Nicotiana benthamiana and served as a positive control. The GFP was used as negative controls in N.benthamiana and tomatoes. Photographs of tobacco were taken 4 days and tomatoes 6 days after infiltration.

3.3.PnSCR82 localizes in the plasma membrane of N.benthamiana leaves

Elicitors,also called apoplastic effectors,are thought to be located in the apoplastic space between the plant cell wall and the plasma membrane. Localization experiments were performed to investigate whether PnSCR82 was localized in the plasma membrane. The transiently expressed PnSCR82-GFP protein signals inN.benthamianaleaves due to agroinfiltration were detected using confocal microscopy at 2 dpa. The green fluorescent protein(GFP) was mainly located within the plasma membrane,cytoplasm and nucleus,although fluorescence was seen throughout the entire cell. In the leaves,the GFP signals of transiently expressed PnSCR82 proteins tagged with GFP mainly accumulated on the plasma membrane(Fig.4-A). To further clarify whether the PnSCR82 was localized in the plasma membrane or the apoplast,the cells were subjected to plasmolysis treatment with 15%NaCl. Cells that expressed GFP alone fluoresced in the cytoplasm while the transiently expressed PnSCR82-GFP accumulated in the plasma membrane (Fig.4-B),indicating that PnSCR82 was localized in the plasma membrane.

Fig.4 PnSCR82 localized in the plasma membrane of Nicotiana benthamiana leaves. A,GFP-tagged PnSCR82 was expressed by agroinfiltration in 4-to 5-week-old N.benthamiana leaves. Bar=75 μm. B,confocal images of plasmolysed tobacco cells expressing PnSCR82 and GFP using 15% NaCl. White arrowheads indicate the plant plasma membrane where it is pulled away from the cell wall. Photographs were taken 48 h after agroinfiltration.

3.4.Manipulation of plant resistance to plant pathogens by PnSCR82

To conclusively verify whether PnSCR82 is involved in manipulating plant resistance,N.benthamianaleaves were exposed to the hemibiotrophic pathogen,P.capsici,24 h after expressingPnSCR82genes.Nicotiana benthamianaleaves infected by these pathogens display visible disease symptoms within 2 days. ThePnSCR82genes and empty vector were infiltrated intoN.benthamianaleaves 24 h prior to infection with the pathogen. GFP construct as the control was transiently expressed on one leaf half,and the other leaf was infiltrated with a candidatePnscr82gene. After 2 days post-inoculation,the disease symptoms were observed on the leaves. Compared with the GFP control,the water-soaked lesions on the expression ofPnSCR82in tobacco leaves reduced dramatically after inoculation withP.capsici. This indicates that leaves expressingPnSCR82showed significantly enhanced resistance toP.capsici(Fig.5-A and B). In addition,the biomass of the host and pathogen DNA was measured by qPCR measurements,with the results showing significant differences between GFP and PnSCR82 (Fig.5-C).Overall,these results confirm PnSCR82’s ability to enhance the immune response in tobacco againstP.capsici.

Fig.5 PnSCR82 enhances the resistance to Phytophthora capsici in Nicotiana benthamiana. A,pathogens were inoculated in the infiltration area 24 h after agroinfiltration. B,the lesion diameter was measured after 2 days post-inoculation (dpi). C,biomass was determined using the ratios of pathogens and taobacco DNA after 2 dpi. Data are mean±SE (n=3).＊,statistical significance between PnSCR82 and GFP (P<0.05).

3.5.PnSCR82 induces plant immune responses in N.benthamiana

To further investigate the importance of PnSCR82 in plant immune responses,the production of ROS and deposition of callose were examined using DAB and aniline blue stains. The deposition of callose inN.benthamianaleaves was shown to be induced by PnSCR82 after 24 h of infiltration,and was significantly increased in plants treated with PnSCR82 after 48 h compared to the GFP control (Fig.6-A). After 24 h of infiltration,we also tested for ROS in the leaves and found that there was no visible ROS accumulation in leaves treated with GFP.A reddish-brown precipitate,however,was observed in leaves treated with PnSCR82 (Fig.6-A),suggesting that the transient expression of PnSCR82 inN.benthamianaleaves induced the plant’s defense responses,especially disease resistance,to thePhytophthorapathogenP.capsici.

To further test alteration of the transcription level of pathogenesis-related genes in PnSCR82-expressing plants,we detected the transcription levels of the defenserelated genes in these signaling pathways. NbPR1 and NbPR2 are involved in SA signal transduction pathway,and probably act as marker genes (Deanet al.2005;Jianget al.2016);NbPR4is a marker gene andNbLOXis an important enzyme gene for JA-dependent defense(Asai and Yoshioka 2009;Kouzaiet al.2016);NbERF1is a ethylene (ET) responsive factor that modulates ethylene response (Preet al.2008;Pieterseet al.2012).Transcriptional levels of the five reference genes were analyzed using qPCR in leaves which had expressed PnSCR82 and GFP 1 to 2 days after agroinfiltration.When compared with the transcript levels of the GFP control,expression ofNbPR2,NbLOXandNbERF1fromN.benthamianaincreased after 24 h agroinfiltration,whereas the expression of NbPR1 was almost unchanged. However,at 48 h agroinfiltration,the expression levels ofNbPR1,NbLOXandNbERF1were up-regulated in the PnSCR82 sample compared to the GFP control. Simultaneously,at 48 h agroinfiltration,the expression levels ofNbPR1,NbPR2andNbPR4were upregulated compared with 24 h agroinfiltration (Fig.6-B).These results suggest that the SA,JA and ET signaling pathways are activated by the transient expression of PnSCR82 inN.benthamianaleaves.

Fig.6 PnSCR82 triggering plant defense responses. A,callose deposition and ROS accumulation in Nicotiana benthamiana leaves after agroinfiltration for 24 or 48 h,the inoculated leaves were then stained with aniline blue and DAB. B,transcription levels of defense-responsive genes in N.benthamiana 24 and 48 h after agroinfiltration. Transcription levels of related genes compared to the endogenous control gene NbEF1α using the comparative Ct method. Data are mean±SD (n=3).＊,statistical significance between PnSCR82 and GFP at P<0.05.

4.Discussion

The PcF toxin family represents a group of small secreted proteins fromPhytophthoraspp. PcF was originally purified fromP.cactorumand was shown to have a toxic effect on the leaves of tomatoes and strawberries,two economically important hosts forP.cactorum(Motteramet al.2009). Apart fromP.cactorum,bioactivity was not verified in other plant pathogenic oomycetes such asP.nicotianae,P.sojae,P.infestans,etc. In the present study,we discovered a small cysteine-rich protein,PnSCR82,fromP.nicotianaeand a member of the PcF toxin family(Fig.1),that induced plant immunity against the oomycete pathogen.

PnSCR82,like PcF toxin family,was up-regulated during the germinating cysts and infectious stages (Fig.2).Specifically,the expression ofSCR96fromP.cactorumwas significantly increased in infectious stages and contributed to its pathogenicity. The PcF-like proteins,SCR91 and SCR74,were identified fromP.infestansand encoded secreted cysteine-rich proteins,that also were significantly up-regulated during plant–pathogen interactions,Birch and Kamoun considered these proteins polymorphic in a key amino acid residue and suggest that the evolution of PcF-like proteins might be related to host–pathogen interreactions (Linet al.2020).

Many fungi andPhytophthoraspecies could deliver small secreted proteins,which can trigger programmed cell death (PCD) when expressed in plant cells(Koomangersmannet al.1997;Slot and Knogge 2002).PCD also plays an important role in providing nutrition for phytopathogens (Dou and Zhou 2012;Mottet al.2014). PnSCR82 could trigger PCD when transiently expressed inN.benthamianaand tomatoes (Fig.3).Interestingly,transient expression of PcSCR82 fromP.capsicitrigger cell death in peppers and tomatoes,but not inN.benthamiana(data not shown). Thus,homologous proteins from different pathogens may have different functions. SCR proteins were not only found in pathogens,but were also confirmed to play important host-defense roles in plants (Tailoret al.1997;Looet al.2017).ToAMP4,a SCR protein,was isolated fromTaraxacum officinaleand displayed broad-spectrum antifungal activity against several phytopathogens(Vermaet al.2012;Astafievaet al.2013).Transgenic expressing Pm-AMP1 in canola demonstrated a reduction in growth of fungal hyphae (Zamanyet al.2011). These data suggest that small cysteine proteins may be an effective and alterable method to protect crops against phytopathogens.

During the hypersensitive response (HR),a plant will rapidly produce the reactive oxygen species (ROS) in an oxidative burst,which then act as signaling molecules to regulate downstream signaling pathways (Carmanet al.1991). ROS could promote plant cell death,kill pathogens directly or indirectly. Furthermore,callose deposits immediately accumulate around an infection by pathogens and limit penetration and growth of pathogens.These reactions are used to quantify the reaction to PAMPs. Our findings demonstrate that the expression of PnSCR82 in tobacco activated plant basal defenses.

It has been reported that the elicitor is localized on the plasma membrane,and is able to recognize special receptor proteins located on plant cell membranes and trigger intracellular defense signalingviathe octadecanoid pathway (Bektas and Eulgem 2014). It is well known that SA,JA,and ET are important signaling networks that mediate plant defense responses against plant pathogens (Kouzaiet al.2016). PR1,PR2,and PR4 were upregulated in the PnSCR82-expressingN.benthamiana,these results are similar to those reported by Zhanget al.(2014) that the expression of defense response-related genes were induced after His-SsCut treatment. The SA signaling pathway is thought to regulate the defense responses against biotrophic plant pathogens,while the JA and ET signaling pathways are responsible for mediating defense responses against necrotrophic plant pathogens (Koornneefet al.2008). HaRxL44 is a nuclear localized effector ofHyaloperonospora arabidopsidisthat enhances plant susceptibility toH.arabidopsidisin transgenic lines ofArabidopsisexpressing HaRxL44,however,Botrytis cinereagrew less well in plant expressing HaRxL44 than in the WT,further analysis of the marker genes from SA,JA and ET pathways found JA/ET responsive genes are activated in HaRxL44-expressing plants simultaneously (Caillaudet al.2013). In VdSCP7-expressing plants,the transcript levels of SA/JA responsive genes (NbPR1,NbPR2,NbLOXandNbERF1)are up-regulated,that explain why VdSCP7 enhanced resistance toB.cinereainfection,but not susceptibility toP.capsiciinfection (Zhanget al.2017). In this case,the SA,JA and ET signaling pathways were involved in the PnSCR82 induced plant defense responses. Therefore,we theorize that PnSCR82 enhanced disease resistance againstP.capsiciin tobacco through the SA signaling pathways.

5.Conclusion

We demonstrated that PnSCR82 induced tobacco basal defense responses such as PCD,ROS,defenserelated protein expression and enhanced plant immunity.Consequently,the possibility of using PnSCR82,or similar proteins,to enhance plant immunity provides an optimistic outlook that utilizing these defense responses may promote plant resistance by replacing or reducing our dependance on agricultural chemicals to control economically important crop pests,and,ultimately,contribute to a healthier environment.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31972218,31501590,and 31601615),the Program for Talents in Qingdao Agricultural University,China (6631114307),and the Independent Innovation of Agricultural Sciences in Jiangsu Province,China (CX(18)3012).

Declaration of competing interest

The authors declare that they have no conflict of interest.