"The PLCP gene family of grapevine (Vitis vinifera L.): characterization and differential expression in response to Plasmopara Viticola".

BACKGROUND: Papain-like cysteine proteases (PLCPs), a large group of cysteine proteases, are structurally related to papain. The members belonging to PLCPs family contribute to plant immunity, senescence, and defense responses in plants. The PLCP gene family has been identified in Arabidopsis, rice, soybean, and cotton. However, no systematic analysis of PLCP genes has been undertaken in grapevine. Since Plasmopara viticola as a destructive pathogen could affect immunity of grapes in the field, we considered that the members belonged to PLCPs family could play a crucial role in defensive mechanisms or programmed cell death. We aimed to evaluate the role of PLCPs in 2 different varieties of grapevines and compared the changes of their expressions with the transcriptional data in response to P. viticola. RESULTS: In this study, 23 grapevine PLCP (VvPLCP) genes were identified by comprehensive bioinformatics analysis. Subsequently, the chromosomal localizations, gene structure, conserved domains, phylogenetic relationship, gene duplication, and cis-acting elements were analyzed. Numerous cis-acting elements related to plant development, hormone, and stress responses were identified in the promoter of the VvPLCP genes. Phylogenetic analysis grouped the VvPLCP genes into nine subgroups. The transcription of VvPLCP in different inoculation time points and varieties indicated that VvPLCP may have vital functions in grapevine defense against Plasmopara viticola. According to transcriptome data and qPCR analysis, we observed the increasing expression levels of VvRD21-1 at 72 h after inoculation in resistant variety, inferring that it was related to grape downy mildew resistance. Meanwhile, 3 genes including VvXBCP1, VvSAG12-1, and VvALP1 showed higher expression at 24 h after pathogen inoculation in the susceptible variety and might be related to the downy mildew phenotype. We nominated these four genes to function during hypersensitive response (HR) process, inferring that these genes could be associated with downy mildew resistance in grapes. CONCLUSIONS: Our results provide the reference for functional studies of PLCP gene family, and highlight its functions in grapevine defense against P. viticola. The results help us to better understand the complexity of the PLCP gene family in plant immunity and provide valuable information for future functional characterization of specific genes in grapevine.

Host apoplastic cysteine protease activity is suppressed during the mutualistic association of Lolium perenne and Epichloë festucae.

Plants secrete various defence-related proteins into the apoplast, including proteases. Papain-like cysteine proteases (PLCPs) are central components of the plant immune system. To overcome plant immunity and successfully colonize their hosts, several plant pathogens secrete effector proteins inhibiting plant PLCPs. We hypothesized that not only pathogens, but also mutualistic microorganisms interfere with PLCP-meditated plant defences to maintain endophytic colonization with their hosts. Epichloë festucae forms mutualistic associations with cool season grasses and produces a range of secondary metabolites that protect the host against herbivores. In this study, we performed a genome-wide identification of Lolium perenne PLCPs, analysed their evolutionary relationship, and classified them into nine PLCP subfamilies. Using activity-based protein profiling, we identified four active PLCPs in the apoplast of L. perenne leaves that are inhibited during endophyte interactions. We characterized the L. perenne cystatin LpCys1 for its inhibitory capacity against ryegrass PLCPs. LpCys1 abundance is not altered during the mutualistic interaction and it mainly inhibits LpCP2. However, since the activity of other L. perenne PLCPs is not sensitive to LpCys1, we propose that additional inhibitors, likely of fungal origin, are involved in the suppression of apoplastic PLCPs during E. festucae infection.

Activity-based proteomics reveals nine target proteases for the recombinant protein-stabilizing inhibitor SlCYS8 in Nicotiana benthamiana.

Co-expression of protease inhibitors like the tomato cystatin SlCYS8 is useful to increase recombinant protein production in plants, but key proteases involved in protein proteolysis are still unknown. Here, we performed activity-based protein profiling to identify proteases that are inhibited by SlCYS8 in agroinfiltrated Nicotiana benthamiana. We discovered that SlCYS8 selectively suppresses papain-like cysteine protease (PLCP) activity in both apoplastic fluids and total leaf extracts, while not affecting vacuolar-processing enzyme and serine hydrolase activity. A robust concentration-dependent inhibition of PLCPs occurred in vitro when purified SlCYS8 was added to leaf extracts, indicating direct cystatin-PLCP interactions. Activity-based proteomics revealed that nine different Cathepsin-L/-F-like PLCPs are strongly inhibited by SlCYS8 in leaves. By contrast, the activity of five other Cathepsin-B/-H-like PLCPs, as well as 87 Ser hydrolases, was unaffected by SlCYS8. SlCYS8 expression prevented protein degradation by inhibiting intermediate and mature isoforms of granulin-containing proteases from the Resistant-to-Desiccation-21 (RD21) PLCP subfamily. Our data underline the key role of endogenous PLCPs on recombinant protein degradation and reveal candidate proteases for depletion strategies.

Papain-like cysteine proteases in Carica papaya: lineage-specific gene duplication and expansion.

BACKGROUND: Papain-like cysteine proteases (PLCPs), a large group of cysteine proteases structurally related to papain, play important roles in plant development, senescence, and defense responses. Papain, the first cysteine protease whose structure was determined by X-ray crystallography, plays a crucial role in protecting papaya from herbivorous insects. Except the four major PLCPs purified and characterized in papaya latex, the rest of the PLCPs in papaya genome are largely unknown. RESULTS: We identified 33 PLCP genes in papaya genome. Phylogenetic analysis clearly separated plant PLCP genes into nine subfamilies. PLCP genes are not equally distributed among the nine subfamilies and the number of PLCPs in each subfamily does not increase or decrease proportionally among the seven selected plant species. Papaya showed clear lineage-specific gene expansion in the subfamily III. Interestingly, all four major PLCPs purified from papaya latex, including papain, chymopapain, glycyl endopeptidase and caricain, were grouped into the lineage-specific expansion branch in the subfamily III. Mapping PLCP genes on chromosomes of five plant species revealed that lineage-specific expansions of PLCP genes were mostly derived from tandem duplications. We estimated divergence time of papaya PLCP genes of subfamily III. The major duplication events leading to lineage-specific expansion of papaya PLCP genes in subfamily III were estimated at 48 MYA, 34 MYA, and 16 MYA. The gene expression patterns of the papaya PLCP genes in different tissues were assessed by transcriptome sequencing and qRT-PCR. Most of the papaya PLCP genes of subfamily III expressed at high levels in leaf and green fruit tissues. CONCLUSIONS: Tandem duplications played the dominant role in affecting copy number of PLCPs in plants. Significant variations in size of the PLCP subfamilies among species may reflect genetic adaptation of plant species to different environments. The lineage-specific expansion of papaya PLCPs of subfamily III might have been promoted by the continuous reciprocal selective effects of herbivore attack and plant defense.

Papain-like cysteine proteases as hubs in plant immunity.

902 I. 902 II. 903 III. 903 IV. 903 V. 905 VI. 905 VII. 905 906 References 906 SUMMARY: Plants deploy a sophisticated immune system to cope with different microbial pathogens and other invaders. Recent research provides an increasing body of evidence for papain-like cysteine proteases (PLCPs) being central hubs in plant immunity. PLCPs are required for full resistance of plants to various pathogens. At the same time, PLCPs are targeted by secreted pathogen effectors to suppress immune responses. Consequently, they are subject to a co-evolutionary host-pathogen arms race. When activated, PLCPs induce a broad spectrum of defense responses including plant cell death. While the important role of PLCPs in plant immunity has become more evident, it remains largely elusive how these enzymes are activated and which signaling pathways are triggered to orchestrate different downstream responses.

Transcriptomic alterations in the sweet orange vasculature correlate with growth repression induced by a variant of citrus tristeza virus.

Citrus tristeza virus (CTV, family Closteroviridae) is an economically important pathogen of citrus. CTV resides in the phloem of the infected plants and induces a range of disease phenotypes, including stem pitting and quick decline as well as a number of other deleterious syndromes. To uncover the biological processes underlying the poorly understood damaging symptoms of CTV, we profiled the transcriptome of sweet orange (Citrus sinensis) phloem-rich bark tissues of non-infected, mock-inoculated trees and trees singly infected with two distinct variants of CTV, T36 or T68-1. The T36 and T68-1 variants accumulated in the infected plants at similar titers. With that, young trees infected with T68-1 were markedly repressed in growth, while the growth rate of the trees infected with T36 was comparable to the mock-inoculated trees. Only a small number of differentially expressed genes (DEGs) were identified in the nearly asymptomatic T36-infected trees, whereas almost fourfold the number of DEGs were identified with the growth-restricting T68-1 infection. DEGs were validated using quantitative reverse transcription-PCR. While T36 did not induce many noteworthy changes, T68-1 altered the expression of numerous host mRNAs encoding proteins within significant biological pathways, including immunity and stress response proteins, papain-like cysteine proteases (PLCPs), cell-wall modifying enzymes, vascular development proteins and others. The transcriptomic alterations in the T68-1-infected trees, in particular, the strong and persistent increase in the expression levels of PLCPs, appear to contribute to the observed stem growth repression. On the other hand, analysis of the viral small interfering RNAs revealed that the host RNA silencing-based response to the infection by T36 and that by T68-1 was comparable, and thus, the induction of this antiviral mechanism may not contribute to the difference in the observed symptoms. The DEGs identified in this study promote our understanding of the underlying mechanisms of the yet unexplained growth repression induced by severe CTV isolates in sweet orange trees.

Hydrogen bonding penalty used for virtual screening to discover potent inhibitors for Papain-Like cysteine proteases of SARS-CoV-2.

The Papain-Like proteases (PLpro) of SARS-CoV-2 play a crucial role in viral replication and the formation of nonstructural proteins. To find available inhibitors, the 3D structure of PLpro of SARS2 was obtained by homologous modelling, and we used this structure as a target to search for inhibitors through molecular docking and MM/GBSA binding free energy rescoring. A novel hydrogen bonding penalty was applied to the screening process, which meanwhile took desolvation into account. Finally, 61 compounds were acquired and 4 of them with IC50 at micromolar level tested in vitro enzyme activity assay, which includes clinical drugs tegaserod. Considering the importance of crystal water molecules, the 4 compounds were re-docked and considered bound waters in the active site as a part of PLpro. The binding modes of these 4 compounds were further explored with metadynamics simulations. The hits will provide a starting point for future key interactions identified and lead optimization targetting PLpro.

Knockout of a papain-like cysteine protease gene OCP enhances blast resistance in rice.

Papain-like cysteine proteases (PLCPs) play an important role in the immune response of plants. In Arabidopsis, several homologous genes are known to be involved in defending against pathogens. However, the effects of PLCPs on diseases that afflict rice are largely unknown. In this study, we show that a PLCP, an oryzain alpha chain precursor (OCP), the ortholog of the Arabidopsis protease RD21 (responsive to dehydration 21), participates in regulating resistance to blast disease with a shorter lesion length characterizing the knockout lines (ocp-ko), generated via CRISPR/Cas9 technology. OCP was expressed in all rice tissues and mainly located in the cytoplasm. We prove that OCP, featuring cysteine protease activity, interacts with OsRACK1A (receptor for activated C kinase 1) and OsSNAP32 (synaptosome-associated protein of 32 kD) physically in vitro and in vivo, and they co-locate in the rice cytoplasm but cannot form a ternary complex. Many genes related to plant immunity were enriched in the ocp-ko1 line whose expression levels changed significantly. The expression of jasmonic acid (JA) and ethylene (ET) biosynthesis and regulatory genes were up-regulated, while that of auxin efflux transporters was down-regulated in ocp-ko1. Therefore, OCP negatively regulates blast resistance in rice by interacting with OsRACK1A or OsSNAP32 and influencing the expression profiles of many resistance-related genes. Moreover, OCP might be the cornerstone of blast resistance by suppressing the activation of JA and ET signaling pathways as well as promoting auxin signaling pathways. Our research provides a comprehensive resource of PLCPs for rice plants in defense against pathogens that is also of potential breeding value.

The Potyviral Protein 6K1 Reduces Plant Proteases Activity during Turnip mosaic virus Infection.

Potyviral genomes encode just 11 major proteins and multifunctionality is associated with most of these proteins at different stages of the virus infection cycle. Some potyviral proteins modulate phytohormones and protein degradation pathways and have either pro- or anti-viral/insect vector functions. Our previous work demonstrated that the potyviral protein 6K1 has an antagonistic effect on vectors when expressed transiently in host plants, suggesting plant defenses are regulated. However, to our knowledge the mechanisms of how 6K1 alters plant defenses and how 6K1 functions are regulated are still limited. Here we show that the 6K1 from Turnip mosaic virus (TuMV) reduces the abundance of transcripts related to jasmonic acid biosynthesis and cysteine protease inhibitors when expressed in Nicotiana benthamiana relative to controls. 6K1 stability increased when cysteine protease activity was inhibited chemically, showing a mechanism to the rapid turnover of 6K1 when expressed in trans. Using RNAseq, qRT-PCR, and enzymatic assays, we demonstrate TuMV reprograms plant protein degradation pathways on the transcriptional level and increases 6K1 stability at later stages in the infection process. Moreover, we show 6K1 decreases plant protease activity in infected plants and increases TuMV accumulation in systemic leaves compared to controls. These results suggest 6K1 has a pro-viral function in addition to the anti-insect vector function we observed previously. Although the host targets of 6K1 and the impacts of 6K1-induced changes in protease activity on insect vectors are still unknown, this study enhances our understanding of the complex interactions occurring between plants, potyviruses, and vectors.

A clubroot pathogen effector targets cruciferous cysteine proteases to suppress plant immunity.

Plant pathogen effector proteins are key to pathogen virulence. In susceptible host Brassicas, the clubroot pathogen, Plasmodiophora brassicae, induces the production of nutrient-sink root galls, at the site of infection. Among a list of 32 P. brassiae effector candidates previously reported by our group, we identified SSPbP53 as a putative apoplastic cystatin-like protein highly expressed during the secondary infection. Here we found that SSPbP53 encoding gene is conserved among several P. brassicae pathotypes and that SSPbP53 is an apoplastic protein able to directly interact with and inhibit cruciferous papain-like cysteine proteases (PLCPs), specifically Arabidopsis XYLEM CYSTEINE PEPTIDASE 1 (AtXCP1). The severity of clubroot disease is greatly reduced in the Arabidopsis xcp1 null mutant (AtΔxcp1) after infection with P. brassicae resting spores, indicating that the interaction of P. brassicae SSPbP53 with XCP1 is important to clubroot susceptibility. SSPbP53 is the first cystatin-like effector identified and characterized for a plant pathogenic protist.

A Conserved Microbial Motif 'Traps' Protease Activation in Host Immunity.

A recent study (Misas-Villamil et al., Nat. Commun., 2019) reveals that Pit2, an apoplastic effector of the corn smut fungus Ustilago maydis, contains an embedded motif of 14 amino acids that binds to and inhibits plant cysteine proteases, thereby modulating host immunity. Intriguingly, the inhibitory motif acts by mimicking the protease substrate and is conserved across microbial kingdoms.

Genome-Wide Identification of Papain-Like Cysteine Proteases in Gossypium hirsutum and Functional Characterization in Response to Verticillium dahliae.

Cotton, a natural fiber producing crop of huge importance, is often prone to attack of Verticillium dahliae. Papain-like cysteine proteases (PLCPs) constitute a large family in plants and were proposed to involve in plant defense against pathogen attack in a number of studies. However, there is no detailed characterization of PLCP genes in cotton against infection of V. dahliae. In this study, we carried out a genome-wide analysis in cotton and identified seventy-eight PLCPs, which were divided into nine subfamilies based on their evolution phylogeny: RD21 (responsive to desiccation 21), CEP (cysteine endopeptidase), XCP (xylem cysteine peptidase), XBCP3 (xylem bark cysteine peptidase 3), THI, SAG12 (senescence-associated gene 12), RD19 (responsive to desiccation 19), ALP (aleurain-like protease) and CTB (cathepsin B-like). Genes in each subfamily exhibit a similar structure and motif composition. The expression patterns of these genes in different organs were examined, and subfamily RD21 was the most abundant in these families. Expression profiles under abiotic stress showed that thirty-five PLCP genes were induced by multiple stresses. Further transcriptome analysis showed that sixteen PLCP genes were up-regulated in response to V. dahliae in cotton. Among those, GhRD21-7 showed a higher transcription level than most other PLCP genes. Additionally, over-expression of GhRD21-7 led to enhanced resistance and RNAi lines were more susceptible to V. dahliae in cotton. Our results provide valuable information for future functional genomic studies of PLCP gene family in cotton.

Sphingolipid-induced cell death in Arabidopsis is negatively regulated by the papain-like cysteine protease RD21.

It is now well established that sphingoid Long Chain Bases (LCBs) are crucial mediators of programmed cell death. In plants, the mycotoxin fumonisin B1 (FB1) produced by the necrotrophic fungus Fusarium moniliforme disrupts the sphingolipid biosynthesis pathway by inhibiting the ceramide synthase leading to an increase in the amount of phytosphingosine (PHS) and dihydrosphingosine (DHS), the two major LCBs in Arabidopsis thaliana. To date, the signaling pathway involved in FB1-induced cell death remains largely uncharacterized. It is also well acknowledged that plant proteases such as papain-like cysteine protease are largely involved in plant immunity. Here, we show that the papain-like cysteine protease RD21 (responsive-to-desiccation-21) is activated in response to PHS and FB1 in Arabidopsis cultured cells and leaves, respectively. Using two allelic null mutants of RD21, and two different PCD bioassays, we demonstrate that the protein acts as a negative regulator of FB1-induced cell death in Arabidopsis.

Role of Papain-Like Cysteine Proteases in Plant Development.

Papain-like cysteine proteases (PLCP) are prominent peptidases found in most living organisms. In plants, PLCPs was divided into nine subgroups based on functional and structural characterization. They are key enzymes in protein proteolysis and involved in numerous physiological processes. In this paper, we reviewed the updated achievements of physiological roles of plant PLCPs in germination, development, senescence, immunity, and stress responses.

Structural characterization of the hypothetical protein Lpg2622, a new member of the C1 family peptidases from Legionella pneumophila.

The Legionella pneumophila type II secretion system can promote bacterial growth under a wide variety of conditions and mediates the secretion of more than 25 proteins, including the uncharacterized effector Lpg2622. Here, we determined the crystal structures of apo-Lpg2622 and Lpg2622 in complex with the cysteine protease inhibitor E64. Structural analysis suggests that Lpg2622 belongs to the C1 family peptidases. Interestingly, unlike the other structurally resolved papain-like cysteine proteases, the propeptide of Lpg2622 forms a novel super-secondary structural fold (hairpin-turn-helix) and can be categorized into a new group. In addition, the N-terminal ß-sheet of the Lpg2622 propeptide plays a regulatory role on enzymatic activity. This study enhances our understanding of the classification and regulatory mechanisms of the C1 family peptidases.

Cytotoxic and molecular impacts of allelopathic effects of leaf residues of Eucalyptus globulus on soybean (Glycine max).

Eucalyptus trees litter plays a crucial role in structuring plant populations and regulating crop quality. To help characterize the allelopathic impact of Eucalyptus plantations and understand the interactions between tree litter and understorey plant populations, we performed two different genomic approaches to determine soybean (Glycine max) crop plant response to biotic stress induced by leaf residues of Eucalyptus globulus trees. For assessing cell death, a qualitative method of DNA fragmentation test (comet assay) was employed to detect cleavage of the genomic DNA into oligonucleosomal fragments and help to characterize the apoptotic event among the experimental samples. In addition, quantitative method of genome analysis at the transcriptional level also was conducted to investigate the expression responses of soybean genome to allelochemicals. Expression of specific genes, which are responsible for the breakdown of proteins during programmed cell death PCD (cysteine proteases and their inhibitors), was examined using semi-quantitative RT-PCR (sqPCR). Results of both conducted analyses proved significant genetic effects of Eucalyptus leaf residues on soybean crop genome, revealed by steady increase in DNA damage as well as variation in the transcript levels of cysteine proteases and inhibitors. Further detailed studies using more sensitive methods are necessary for a comprehensive understanding of the allelopathic effects of Eucalyptus plantations on crops.

Identification and characterization of MOR-CP, a cysteine protease induced by ozone and developmental senescence in maize (Zea mays L.) leaves.

Among the different classes of endoproteases, cysteine proteases are consistently associated with senescence, defense signaling pathways and cellular responses to abiotic stresses. The objectives of this work were to study the effects of various concentrations of ozone on gene expression and enzymatic activity for papain-like cysteine proteases (PLCPs), in the leaves of maize plants grown under field conditions. Leaves from ranks 12 and 10 (cob leaf) were harvested regularly over a long-term artificial ozone fumigation experiment (50 d). Tissues were tested for transcriptional and activity changes concerning cysteine proteases, using qRT-PCR for the newly identified ozone-responsive PLCP gene (Mor-CP) and synthetic oligopeptide Boc-Val-Leu-Lys-AMC as a PLCP-specific substrate, respectively. Results showed that developmental senescence induced a significant and progressive rise in CP activity, only in the older leaves 10 and had no effect on Mor-CP gene expression levels. On the other hand, ozone dramatically enhanced Mor-CP mRNA levels and global PLCP enzymatic activity in leaves 12 and 10, particularly toward the end of the treatment. Ozone impact was more pronounced in the older leaves 10. Together, these observations concurred to conclude that ozone stress enhances natural senescence processes, such as those related to proteolysis.

E-64c-hydrazide: a lead structure for the development of irreversible cathepsin†C inhibitors.

Cathepsin C is a papain-like cysteine protease with dipeptidyl aminopeptidase activity that is thought to activate various granule-associated serine proteases. Its exopeptidase activity is structurally explained by the so-called exclusion domain, which blocks the active-site cleft beyond the S2 site and, with its Asp 1 residue, provides an anchoring point for the N terminus of peptide and protein substrates. Here, the hydrazide of (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane (E-64c) (k2/Ki =140±5 M(-1) s(-1)) is demonstrated to be a lead structure for the development of irreversible cathepsin C inhibitors. The distal amino group of the hydrazide moiety addresses the acidic Asp 1 residue at the entrance of the S2 pocket by hydrogen bonding while also occupying the flat hydrophobic S1'-S2' area with its leucine-isoamylamide moiety. Furthermore, structure-activity relationship studies revealed that functionalization of this distal amino group with alkyl residues can be used to occupy the conserved hydrophobic S2 pocket. In particular, the n-butyl derivative was identified as the most potent inhibitor of the series (k2/Ki =56 000±1700 M(-1) s(-1)).