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Over the past decades, numerous studies have demonstrated that proteases serve as a crucial regulatory mechanism in controlling plant immunity. In this review, we specifically focus on the role of one subfamily of RD21-like papain-like cysteine proteases that carry a C-terminal granulin domain. These proteases share high homology but have been described under very different names in different plant species. We provide a comprehensive overview of the background, endogenous regulation, and subcellular localization of RD21-like proteases in plants. Notably, RD21-like proteases act in immunity against various pathogens and they are targeted by many unrelated pathogen-secreted effectors that inactivate, mislocalise or degrade RD21-like proteases. We highlight open questions and strategies to use this knowledge to develop innovative strategies for crop protection., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Cysteine protease from grapevine (Vitis vinifera) belongs to those resistant proteins, which survive the process of vinification and can therefore be detected as wine components. Its amino acid sequence shows a homology to other members of the papain family, but the enzyme has only partially been explored so far. In order to get more biochemical information with the help of mass spectrometry (MS), wine proteins were collected by ultrafiltration and separated by gel permeation chromatography. The purified enzyme surprisingly displayed a high molecular mass value of around 200 kDa, indicating a possible oligomeric status and aggregation, as it entered only negligibly the separating 10% gel during polyacrylamide gel electrophoresis. The isoelectric point (pI) value of 3.6 was determined by chromatofocusing. Matrix-assisted laser desorption/ionization (MALDI)-MS was employed to evaluate the cleavage specificity and usefulness of the isolated cysteine protease in protein and peptide research. A potential applicability could be anticipated from the efficient digestion performance in volatile ammonium formate buffers at pH 3. Common peptides were digested and the resulting products analyzed by MS/MS sequencing. Then, mixtures of protein standards and extracted barley nuclear proteins were processed in the same way. Grape cysteine protease is nonspecific but shows a certain preference for Arg, Lys, and also Leu residues. Compared with papain, it seems not to require fully the presence of a large hydrophobic residue adjacent to that at the cleavage site. The enzyme is suitable for protein research as it produces peptides of a reasonable length in acidic pH. [ABSTRACT FROM AUTHOR]

Highlights • RD21 is activated in Arabidopis cultured cell in response to PHS. • RD21 is activated in Arabidopsis leaves in response to fumonisin B1. • RD21 behaves as a negative regulator of sphingolipid-induced cell death. Abstract 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. [ABSTRACT FROM AUTHOR]

Autophagy is essential for protein degradation, nutrient recycling, and nitrogen remobilization. Autophagy is induced during leaf ageing and in response to nitrogen starvation, and is known to play a fundamental role in nutrient recycling for remobilization and seed filling. Accordingly, ageing leaves of Arabidopsis autophagy mutants (atg) have been shown to over-accumulate proteins and peptides, possibly because of a reduced protein degradation capacity. Surprisingly, atg leaves also displayed higher protease activities. The work reported here aimed at identifying the nature of the proteases and protease activities that accumulated differentially (higher or lower) in the atg mutants. Protease identification was performed using shotgun LC-MS/MS proteome analyses and activity-based protein profiling (ABPP). The results showed that the chloroplast FTSH (FILAMENTATION TEMPERATURE SENSITIVE H) and DEG (DEGRADATION OF PERIPLASMIC PROTEINS) proteases and several extracellular serine proteases [subtilases (SBTs) and serine carboxypeptidase-like (SCPL) proteases] were less abundant in atg5 mutants. By contrast, proteasomerelated proteins and cytosolic or vacuole cysteine proteases were more abundant in atg5 mutants. Rubisco degradation assays and ABPP showed that the activities of proteasome and papain-like cysteine protease were increased in atg5 mutants. Whether these proteases play a back-up role in nutrient recycling and remobilization in atg mutants or act to promote cell death is discussed in relation to their accumulation patterns in the atg5 mutant compared with the salicylic acid-depleted atg5/sid2 double-mutant, and in low nitrate compared with high nitrate conditions. Several of the proteins identified are indeed known as senescence- and stress-related proteases or as spontaneous cell-death triggering factors. [ABSTRACT FROM AUTHOR]

Proteolytic enzymes (proteases) participate in a vast range of physiological processes, ranging from nutrient digestion to blood coagulation, thrombosis, and beyond. In plants, proteases are implicated in host recognition and pathogen infection, induced defense (immunity), and the deterrence of insect pests. Because proteases irreversibly cleave peptide bonds of protein substrates, their activity must be tightly controlled in time and space. Here, we report an example of how nature evolved alternative mechanisms to fine-tune the activity of a cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION-21). One mechanism in the model plant Arabidopsis thaliana studied here comprises irreversible inhibition of RD21’s activity by Serpin1, whereas the other mechanism is a result of the reversible inhibition of RD21 activity by a Kunitz protease inhibitor named water-soluble chlorophyll-binding protein (WSCP). Activity profiling, complex isolation, and homology modeling data revealed unique interactions of RD21 with Serpin1 and WSCP, respectively. Expression studies identified only partial overlaps in Serpin1 and WSCP accumulation that explain how RD21 contributes to the innate immunity of mature plants and arthropod deterrence of seedlings undergoing skotomorphogenesis and greening. [ABSTRACT FROM AUTHOR]

RD21 (Responsive to desiccation-21) is an Arabidopsis cysteine protease which possesses a granulin-like domain at the C-terminus. Although two forms of RD21 have been identified, consisting of an intermediate form (iRD21) containing a granulin domain and a mature form (mRD21) lacking this domain, the enzymatic properties of these enzymes remain poorly understood. In this study, mRD21 orthologue was purified to homogeneity from the cotyledons of daikon radish (Raphanus sativus). RD21 preferentially cleaved peptide bond that had an aromatic or hydrophobic amino acid at the P2 position. Furthermore, the presence of a polar amino acid at the P1 position enhanced the cleavage susceptibility of the peptide bond, although the importance of the type of amino acid residue at the P1 and P1′ positions was not as significant as the residue located at the P2 position. The iRD21 was also identified as an oligomeric form by gel filtration and sedimentation analyses. The expression of RD21 mRNA was initiated by imbibition and continued at almost constant levels during germination. On the other hand, the enzyme activity increased markedly 5 days after imbibition. These results indicate that this elevation of RD21 activity is generated post-transcriptionally. [ABSTRACT FROM PUBLISHER]