Papain-like Cysteine Proteases Research Articles

Papain-like cysteine protease gene family in fig (Ficus carica L.): genome-wide analysis and expression patterns

Papain-like cysteine protease gene family in fig (<i>Ficus carica</i> L.): genome-wide analysis and expression patterns

Genetic trans-complementation of L-protease fails to rescue the infectious foot-and-mouth disease virus from the Lbpro defective genome

Outbreaks of the foot-and-mouth disease (FMD) have major economic impact on the global livestock industry by affecting the animal health and product safety. L-protease, a non-structural protein of FMDV, is a papain-like cysteine proteinase involved in viral protein processing as well as cleavage of host proteins for promoting the virus growth. FMDV synthesizes two forms of leader proteinase, Lpro (Labpro and Lbpro), where the deletion of Labpro is lethal and Lbpro deletion is reported to be attenuated. Defective replicons have been used by trans-complementing the deleted gene to produce one time replicating virus; thus, the bio-safety procedure can be compromised in the production units. Attempts are made to rescue of ΔLbproFMDV Asia1 virus by co-expressing the Lbpro protein carried in pcDNA plasmid. Mutant FMDV cDNA, pAsia-ΔLbpro, was constructed by PCR mediated mutagenesis using inverse primers. Transfection of BHK-21 cells with in-vitro transcribed RNA from the constructs failed to produce an infective mutant FMDV. Genetic trans-complementation of the Lbpro, which was done by co-transfecting the pcDNALbpro plasmid DNA along with the pAsia-ΔLbpro RNA in BHK-21 cells also failed to produce viable virus. Expression experiments of reporter genes and indirect immune-fluorescence confirmed the production of the viral proteins in wild type FMDV pAsiaWT; however, it was absent in the pAsia-ΔLbpro indicating that the leaderless virus was unable to produce infectious progeny and infect the cells. Failure to produce virus either by Lbpro deleted mutant clone or by genetic complementation suggests little chance of reversion of the disabled virus with large deletions of FMDV genome.

Genome-Wide Identification and Male Sterility-Related Expression Analysis of Papain-like Cysteine Protease Gene Family in Capsicum annuum

PLCPs (papain-like cysteine proteases) are one of the most abundant groups of cysteine proteases and play vital roles in multiple processes. The pepper (Capsicum annuum) is an important Solanaceae vegetable crop; its commercial hybrid seeds are widely used in production. Male sterility is a valuable trait for hybrid seed production. However, the function of PLCPs and the underlying mechanisms of male sterility in peppers remain unclear. In this study, we comprehensively identified the PLCP gene family in peppers, identifying 31 CaPLCPs. A phylogenetic analysis classified 31 members into eight clades. These CaPLCPs were unevenly distributed across eight chromosomes, and five segmental duplicated pairs were observed. The promoter cis-acting element analysis indicated that CaPLCP promoters contained abundant hormone-responsive and stress-responsive cis-elements, suggesting that CaPLCPs may play important roles in responding to abiotic stress, such as drought and low temperatures, as well as in plant immunity. The qRT-PCR analysis demonstrated that the expression levels of CaPLCP1, CaPLCP5, CaPLCP11, CaPLCP12, CaPLCP13, CaPLCP17, CaPLCP19, and CaPLCP21 were significantly reduced in the flowers of MS (male sterile pepper) at least at one stage, indicating their potential roles as regulatory factors in pepper male sterility. These findings provide important insights into the functional analysis of the PLCP gene family in peppers and other species, laying a crucial foundation for understanding the mechanisms of male sterility in peppers.

Noscapine shows antimalarial activity against Plasmodium falciparum 3D7, its clinical isolate Pf140/SS, and Plasmodium berghei ANKA

Objective: To evaluate the antimalarial activity of noscapine against Plasmodium falciparum 3D7 strain (Pf3D7), its clinical isolate (Pf140/SS), and Plasmodium berghei ANKA (PbA). Methods: Using ring-stage survival assay, phenotypic assessments, and SYBR-green-based fluorescence assay, the antimalarial activities of noscapine were assessed compared with dihydroartemisinin (DHA) in in vivo and in vitro studies. In addition, hemolysis and cytotoxicity tests were carried out to evaluate its safety. RT-PCR assay was also conducted to determine the effect of noscapine on papain-like cysteine protease Plasmodium falciparum falcipain-2 (PfFP-2). Results: The antimalarial efficacy of noscapine against Pf3D7 and Pf140/SS was comparable to DHA, with IC50 values of (7.68±0.88) and (5.57±0.74) nM/mL, respectively, and &gt;95% inhibition of PbA infected rats. Noscapine also showed a safe profile, as evidenced by low hemolysis and cytotoxicity even at high concentrations. Moreover, PfFP-2 expression was significantly inhibited in both noscapine-treated Pf3D7 and Pf140/SS (P&lt;0.01). Conclusions: Noscapine has antimalarial properties comparable to standard antimalarial DHA with better safety profiles, which may be further explored as a therapeutic candidate for the treatment of malaria.

The salivary gland transcriptome of Varroa destructor reveals suitable targets for RNAi-based mite control.

The mite Varroa destructorAnderson and Trueman (Mesostigmata: Varroidae) has a dramatic impact on beekeeping and is one of the main causes of honey bee colony losses. This ectoparasite feeds on honey bees' liquid tissues, through a wound created on the host integument, determining weight loss and a reduction of lifespan, as well as the transmission of viral pathogens. However, despite its importance, the mite feeding strategy and the host regulation role by the salivary secretions have been poorly explored. Here, we contribute to fill this gap by identifying the salivary components of V. destructor, to study their functional importance for mite feeding and survival. The differential expression analysis identified 30 salivary gland genes encoding putatively secreted proteins, among which only 15 were found to be functionally annotated. These latter include proteins with putative anti-bacterial, anti-fungal, cytolytic, digestive and immunosuppressive function. The three most highly transcribed genes, coding for a chitin-binding domain protein, a Kazal domain serine protease inhibitor and a papain-like cysteine protease were selected to study their functional importance by reverse genetics. Knockdown (90%-99%) by RNA interference (RNAi) of the transcript of a chitin-binding domain protein, likely interfering with the immune reaction to facilitate mite feeding, was associated with a 40%-50% decrease of mite survival. This work expands our knowledge of the host regulation and nutritional exploitation strategies adopted by ectoparasites of arthropods and allows the identification of potential targets for RNAi, paving the way towards the development of new strategies for Varroa mite control.

Unveiling the Roles of Cysteine Proteinases F and W: From Structure to Pathological Implications and Therapeutic Targets.

Cysteine cathepsins F and W are members of the papain-like cysteine protease family, which have distinct structural features and functional roles in various physiological and pathological processes. This review provides a comprehensive overview of the current understanding of the structure, biological functions, and pathological implications of cathepsins F and W. Beginning with an introduction to these proteases, we delve into their structural characteristics and elucidate their unique features that dictate their enzymatic activities and substrate specificity. We also explore the intricate involvement of cathepsins F and W in malignancies, highlighting their role as potential biomarkers and therapeutic targets in cancer progression. Furthermore, we discuss the emerging roles of these enzymes in immune response modulation and neurological disorders, shedding light on their implications in autoimmune and neurodegenerative diseases. Finally, we review the landscape of inhibitors targeting these proteases, highlighting their therapeutic potential and challenges in clinical translation. This review brings together the diverse facets of cysteine cathepsins F and W, providing insights into their roles in health and disease and guiding future investigations for therapeutic advances.

Mechanistic insights into CrCEP1: A dual-function cysteine protease with endo- and transpeptidase activity

Proteases, essential regulators of plant stress responses, remain enigmatic in their precise functional roles. By employing activity-based probes for real-time monitoring, this study aimed to delve into protease activities in Chlamydomonas reinhardtii exposed to oxidative stress induced by hydrogen peroxide. However, our work revealed that the activity-based probes strongly labelled three non-proteolytic proteins—PsbO, PsbP, and PsbQ—integral components of photosystem II's oxygen-evolving complex. Subsequent biochemical assays and mass spectrometry experiments revealed the involvement of CrCEP1, a previously uncharacterized papain-like cysteine protease, as the catalyst of this labelling reaction. Further experiments with recombinant CrCEP1 and PsbO proteins replicated the reaction in vitro. Our data unveiled that endopeptidase CrCEP1 also has transpeptidase activity, ligating probes and peptides to the N-termini of Psb proteins, thereby expanding the repertoire of its enzymatic activities. The hitherto unknown transpeptidase activity of CrCEP1, working in conjunction with its proteolytic activity, unveils putative complex and versatile roles for proteases in cellular processes during stress responses.

Proximity Labelling Confirms the Involvement of Papain-Like Cysteine Proteases and Chaperones in Cyclotide Biosynthesis

Cyclotides are disulfide-rich insecticidal peptides from plants that harbour a cyclic cystine knot motif that imparts them with high stability. Like most ribosomally encoded peptides, cyclotides are produced as precursor proteins that are subsequently processed to maturity through proteolytic activity. Although the final maturation of the prototypic cyclotide kalata B1 (kB1) is known to involve asparaginyl endopeptidases, the role of the in planta folding machinery is less well characterized, as is also the case for in planta N-terminal processing enzyme(s). Here, we used proximity labelling to identify proteins involved in the biosynthesis of kB1 in both a cyclotide-bearing plant species, Petunia × hybrida [Regel], and in Nicotiana benthamiana [Domin] that does not naturally produce cyclotides. Together, several ER resident chaperones, protein disulfide isomerases, a papain-like cysteine protease, and an asparaginyl endopeptidase were identified in the kB1 interactome. We found that overexpression of the identified papain-like cysteine protease resulted in an improvement in the yield of cyclic kB1, while this was not the case for the overexpression of protein disulfide isomerases, despite their interaction being verified using bimolecular fluorescence complementation. Together, these results provide a significant advance in our understanding of the role of auxiliary biosynthetic elements for heterologous cyclotide production in plants.

NIa-Pro of sugarcane mosaic virus targets Corn Cysteine Protease 1 (CCP1) to undermine salicylic acid-mediated defense in maize.

Papain-like cysteine proteases (PLCPs) play pivotal roles in plant defense against pathogen invasions. While pathogens can secrete effectors to target and inhibit PLCP activities, the roles of PLCPs in plant-virus interactions and the mechanisms through which viruses neutralize PLCP activities remain largely uncharted. Here, we demonstrate that the expression and activity of a maize PLCP CCP1 (Corn Cysteine Protease), is upregulated following sugarcane mosaic virus (SCMV) infection. Transient silencing of CCP1 led to a reduction in PLCP activities, thereby promoting SCMV infection in maize. Furthermore, the knockdown of CCP1 resulted in diminished salicylic acid (SA) levels and suppressed expression of SA-responsive pathogenesis-related genes. This suggests that CCP1 plays a role in modulating the SA signaling pathway. Interestingly, NIa-Pro, the primary protease of SCMV, was found to interact with CCP1, subsequently inhibiting its protease activity. A specific motif within NIa-Pro termed the inhibitor motif was identified as essential for its interaction with CCP1 and the suppression of its activity. We have also discovered that the key amino acids responsible for the interaction between NIa-Pro and CCP1 are crucial for the virulence of SCMV. In conclusion, our findings offer compelling evidence that SCMV undermines maize defense mechanisms through the interaction of NIa-Pro with CCP1. Together, these findings shed a new light on the mechanism(s) controlling the arms races between virus and plant.

Plant and Arthropod IgE-Binding Papain-like Cysteine Proteases: Multiple Contributions to Allergenicity.

Papain-like cysteine proteases are widespread and can be detected in all domains of life. They share structural and enzymatic properties with the group's namesake member, papain. They show a broad range of protein substrates and are involved in several biological processes. These proteases are widely exploited for food, pharmaceutical, chemical and cosmetic biotechnological applications. However, some of them are known to cause allergic reactions. In this context, the objective of this review is to report an overview of some general properties of papain-like cysteine proteases and to highlight their contributions to allergy reactions observed in humans. For instance, the literature shows that their proteolytic activity can cause an increase in tissue permeability, which favours the crossing of allergens through the skin, intestinal and respiratory barriers. The observation that allergy to PLCPs is mostly detected for inhaled proteins is in line with the reports describing mite homologs, such as Der p 1 and Der f 1, as major allergens showing a frequent correlation between sensitisation and clinical allergic reactions. In contrast, the plant food homologs are often digested in the gastrointestinal tract. Therefore, they only rarely can cause allergic reactions in humans. Accordingly, they are reported mainly as a cause of occupational diseases.

Targeting Magnaporthe oryzae effector MoErs1 and host papain-like protease OsRD21 interaction to combat rice blast.

Effector proteins secreted by plant pathogenic fungi are important artilleries against host immunity, but there is no precedent of such effectors being explored as antifungal targets. Here we demonstrate that MoErs1, a species-specific effector protein secreted by the rice blast fungus Magnaporthe oryzae, inhibits the function of rice papain-like cysteine protease OsRD21 involved in rice immunity. Disrupting MoErs1-OsRD21 interaction effectively controls rice blast. In addition, we show that FY21001, a structure-function-based designer compound, specifically binds to and inhibits MoErs1 function. FY21001 significantly and effectively controls rice blast in field tests. Our study revealed a novel concept of targeting pathogen-specific effector proteins to prevent and manage crop diseases.

Crystal structure and biochemical activity of the macrodomain from rubella virus p150.

Rubella virus encodes a nonstructural polyprotein with RNA polymerase, methyltransferase, and papain-like cysteine protease activities, along with a putative macrodomain of unknown function. Macrodomains bind ADP-ribose adducts, a post-translational modification that plays a key role in host-virus conflicts. Some macrodomains can also remove the mono-ADP-ribose adduct or degrade poly-ADP-ribose chains. Here, we report high-resolution crystal structures of the macrodomain from rubella virus nonstructural protein p150, with and without ADP-ribose binding. The overall fold is most similar to macroD-type macrodomains from various nonviral species. The specific composition and structure of the residues that coordinate ADP-ribose in the rubella virus macrodomain are most similar to those of macrodomains from alphaviruses. Isothermal calorimetry shows that the rubella virus macrodomain binds ADP-ribose in solution. Enzyme assays show that the rubella virus macrodomain can hydrolyze both mono- and poly-ADP-ribose adducts. Site-directed mutagenesis identifies Asn39 and Cys49 required for mono-ADP-ribosylhydrolase (de-MARylation) activity.IMPORTANCERubella virus remains a global health threat. Rubella infections during pregnancy can cause serious congenital pathology, for which no antiviral treatments are available. Our work demonstrates that, like alpha- and coronaviruses, rubiviruses encode a mono-ADP-ribosylhydrolase with a structurally conserved macrodomain fold to counteract MARylation by poly (ADP-ribose) polymerases (PARPs) in the host innate immune response. Our structural data will guide future efforts to develop novel antiviral therapeutics against rubella or infections with related viruses.

Processing of the Hepatitis E virus ORF1 nonstructural polyprotein

Hepatitis E viruses (HEV) Open Reading Frame 1 (ORF1) encodes a non-structural polyprotein. In most positive-sense RNA viruses found in animals, this non-structural polyprotein is cleaved by viral protease or host protease. However, the mechanism behind the processing of HEV polyprotein remains one of the most controversial questions in HEV biology. The role of putative HEV protease in processing is difficult to demonstrate. Recent studies have questioned the existence of HEV protease and suggested that pORF1 lacks protease activity. Conversely, studies also suggested the role of host proteases involved in the blood coagulation cascade, like thrombin, in processing the HEV pORF1 polyprotein. In summary, recent studies support the notion that pORF1 lacks protease activity and host proteases are responsible for processing pORF1. The present review compiles a thorough overview of contentious research on HEV’s papain-like cysteine protease (PCP) and highlights recent advancements in the field. We aim to discuss the challenges and opportunities in the field to focus on further research.

The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal Release

BackgroundA peculiar feature of the hepatitis E virus (HEV) is its reliance on the exosomal route for viral release. Genomic replication is mediated via the viral polyprotein pORF1, yet little is known about its subcellular localization. MethodsSubcellular localization of pORF1 and its subdomains, generated and cloned based on a structural prediciton of the viral replicase, was analyzed via confocal laser scanning microscopy. Exosomes released from cells were isolated via ultracentrifugation and analyzed by isopycnic density gradient centrifugation. This was followed by fluorimetry or Western Blot analyses or RT-qPCR to analyze separated particles in more detail. ResultsWe found pORF1 to be accumulating within the endosomal system, most dominantly to MVBs. Expression of the polyprotein’s seven subdomains revealed that the PCP (papain-like cysteine-protease) is the only domain localizing like the full-length protein. A PCP-deficient pORF1 mutant lost its association to MVBs. Strikingly, both pORF1 and PCP can be released via exosomes. Similarly, genomic RNA still is released via exosomes in the absence of pORF2/3. ConclusionsTaken together we found that pORF1 localizes to MVBs in a PCP-dependent manner, which is followed by exosomal release. This reveals new aspects of HEV life cycle, as replication and release could be coupled at the endosomal interface. Additionally, this may mediate capsid-independent spread or may facilitate the spread of viral infection, as genomes entering the cell during de novo infection readily encounter exosomally-transferred pORF1.

Comparative analysis of cysteine proteases reveals gene family evolution of the group 1 allergens in astigmatic mites.

Astigmatic mites contain potent allergens that can trigger IgE-mediated immune responses, leading to allergic diseases such as asthma, allergic rhinitis and atopic dermatitis. In house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae, group 1 allergens (Der p 1 and Der f 1), characterized as papain-like cysteine proteases, have been defined as the major allergens that have high prevalence and potency. Previous studies of mite group 1 allergens mainly focused on identification, comparison of sequence and structure, as well as the investigation of cross-reactivity. To achieve a comprehensive view of mite group 1 allergens, we performed a comparative genomic analysis of all the cysteine proteases in six astigmatic mite species to elucidate the evolutionary relationships of group 1 allergens. Based on the high-quality and annotated genomes, all the cysteine proteases in six astigmatic mite species were identified by sequence homology search. The phylogenetic relationships, gene synteny and expression levels were revealed by bioinformatic tools. The allergenicity of recombinant cysteine proteases was evaluated by enzyme-linked immunosorbent assay. Tandem duplication was revealed as the major feature of cysteine protease gene evolution in astigmatic mites. The high IgE-binding capacity and the significant expression level of the cysteine protease DP_007902.01 suggested its potential as a novel group 1 allergen of D. pteronyssinus. In addition, gene decay events were identified in the skin-burrowing parasitic mite Sarcoptes scabiei. This comprehensive analysis provided insights into the evolution of cysteine proteases, as well as the component-resolved diagnosis of mite allergies.

A papain-like cysteine protease-released small signal peptide confers wheat resistance to wheat yellow mosaic virus

Wheat yellow mosaic virus (WYMV), a soil-borne pathogen, poses a serious threat to global wheat production. Here, we identify a WYMV resistance gene, TaRD21A, that belongs to the papain-like cysteine protease family. Through genetic manipulation of TaRD21A expression, we establish its positive role in the regulation of wheat to WYMV resistance. Furthermore, our investigation shows that the TaRD21A-mediated plant antiviral response relies on the release of a small peptide catalyzed by TaRD21A protease activity. To counteract wheat resistance, WYMV-encoded nuclear inclusion protease-a (NIa) suppress TaRD21A activity to promote virus infection. In resistant cultivars, a natural variant of TaRD21A features a alanine to serine substitution and this substitution enables the phosphorylation of Serine, thereby weakening the interaction between NIa and TaRD21A, reinforcing wheat resistance against WYMV. Our study not only unveils a WYMV resistance gene but also offers insights into the intricate mechanisms underpinning resistance against WYMV.

Methotrexate, an anti-inflammatory drug, inhibits Hepatitis E viral replication

Hepatitis E Virus (HEV) is a positively oriented RNA virus having a 7.2 kb genome. HEV consists of three open reading frames (ORF1-3). Of these, ORF1 codes for the enzymes Methyltransferase (Mtase), Papain-like cysteine protease (PCP), RNA helicase, and RNA-dependent RNA polymerase (RdRp). Unavailability of a vaccine or effective drug against HEV and considering the side effects associated with the off-label use of ribavirin (RBV) and pegylated interferons, an alternative approach is required by the modulation of specific enzymes to prevent the infection. HEV helicase is involved in unwinding the double-stranded RNA, RNA processing, transcriptional regulation, and pre-mRNA processing. Therefore, we screened FDA-approved compounds from the ZINC15 database against the modelled 3D structure of HEV helicase and found that methotrexate and compound A (Pubchem ID BTB07890) inhibit the NTPase and dsRNA unwinding activity leading to inhibition of HEV RNA replication. This may be further authenticated by in vivo study.

The Role of Cysteine Protease Cathepsins B, H, C, and X/Z in Neurodegenerative Diseases and Cancer.

Papain-like cysteine proteases are composed of 11 human cysteine cathepsins, originally located in the lysosomes. They exhibit broad specificity and act as endopeptidases and/or exopeptidases. Among them, only cathepsins B, H, C, and X/Z exhibit exopeptidase activity. Recently, cysteine cathepsins have been found to be present outside the lysosomes and often participate in various pathological processes. Hence, they have been considered key signalling molecules. Their potentially hazardous proteolytic activities are tightly regulated. This review aims to discuss recent advances in understanding the structural aspects of these four cathepsins, mechanisms of their zymogen activation, regulation of their activities, and functional aspects of these enzymes in neurodegeneration and cancer. Neurodegenerative effects have been evaluated, particularly in Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, and neuropsychiatric disorders. Cysteine cathepsins also participate in tumour progression and metastasis through the overexpression and secretion of proteases, which trigger extracellular matrix degradation. To our knowledge, this is the first review to provide an in-depth analysis regarding the roles of cysteine cathepsins B, H, C, and X in neurodegenerative diseases and cancer. Further advances in understanding the functions of cysteine cathepsins in these conditions will result in the development of novel, targeted therapeutic strategies.

Molecular characterization and pathogenicity evaluation of enterovirus G isolated from diarrheic piglets.

Four enterovirus G (EV-G) strains were isolated from diarrheic piglets that were negative for common swine enteric viruses. The spherical enterovirus particles of roughly 30-nm diameter were observed under transmission electron microscopy by using plaque-purified enterovirus. The complete genome sequence analysis revealed that each of four enteroviruses contained a papain-like cysteine protease (PLCP) gene between the 2C and 3A junction regions of the viral genome. This insertion encoded a predicted protease similar to the PLCP of porcine torovirus. The phylogenetic analysis based on complete genome with and without PLCP gene revealed that the four isolated EV-G strains were grouped together with global enterovirus G1-PLCP strains, and more closely related to EV-G/PLCP strains previously detected in China, Japan, and Korea (90.3%-92.2% similarities based on nucleotides). The cell susceptibility test demonstrated that the isolated EV-G could infect and replicate in cell lines from various host species. Furthermore, pathogenicity evaluation showed that the isolated EV-Gs induced mild diarrhea, pyrexia, and reduced body weight in infected piglets. The epidemiological investigation revealed a high prevalence of EV-G in swine herds. Together, our findings demonstrate that the isolated EV-G is pathogenic in piglets and may be advantageous in providing more trustworthy data on the evolution and pathological properties of EV-G. IMPORTANCE Enterovirus G is a species of positive-sense single-stranded RNA viruses associated with several mammalian diseases. The porcine enterovirus strains isolated here were chimeric viruses with the PLCP gene of porcine torovirus, which grouped together with global EV-G1 strains. The isolated EV-G strain could infect various cell types from different species, suggesting its potential cross-species infection risk. Animal experiment showed the pathogenic ability of the isolated EV-G to piglets. Additionally, the EV-Gs were widely distributed in the swine herds. Our findings suggest that EV-G may have evolved a novel mechanism for broad tropism, which has important implications for disease control and prevention.

XYLEM CYSTEINE PEPTIDASE 1 and its inhibitor CYSTATIN 6 regulate pattern-triggered immunity by modulating the stability of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D.

Plants produce a burst of reactive oxygen species (ROS) after pathogen infection to successfully activate immune responses. During pattern-triggered immunity (PTI), ROS are primarily generated by the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD). RBOHD is degraded in the resting state to avoid inappropriate ROS production; however, the enzyme mediating RBOHD degradation and how to prevent RBOHD degradation after pathogen infection is unclear. In this study, we identified an Arabidopsis (Arabidopsis thaliana) vacuole-localized papain-like cysteine protease, XYLEM CYSTEINE PEPTIDASE 1 (XCP1), and its inhibitor CYSTATIN 6 (CYS6). Pathogen-associated molecular pattern-induced ROS burst and resistance were enhanced in the xcp1 mutant but were compromised in the cys6 mutant, indicating that XCP1 and CYS6 oppositely regulate PTI responses. Genetic and biochemical analyses revealed that CYS6 interacts with XCP1 and depends on XCP1 to enhance PTI. Further experiments showed that XCP1 interacts with RBOHD and accelerates RBOHD degradation in a vacuole-mediated manner. CYS6 inhibited the protease activity of XCP1 toward RBOHD, which is critical for RBOHD accumulation upon pathogen infection. As CYS6, XCP1, and RBOHD are conserved in all plant species tested, our findings suggest the existence of a conserved strategy to precisely regulate ROS production under different conditions by modulating the stability of RBOHD.