Plants sense the presence of pathogens or pests through the recognition of evolutionarily conserved microbe- or herbivore-associated molecular patterns or specific pathogen effectors, as well as plant endogenous danger-associated molecular patterns. This sensory capacity is largely mediated through plasma membrane and cytosol-localized receptors which trigger complex downstream immune signaling cascades. As immune signaling outputs are often associated with a high fitness cost, precise regulation of this signaling is critical. Protease-mediated proteolysis represents an important form of pathway regulation in this context. Proteases have been widely implicated in plant–pathogen interactions, and their biochemical mechanisms and targets continue to be elucidated. During the plant and pathogen arms race, specific proteases are employed from both the plant and the pathogen sides to contribute to either defend or invade. Several pathogen effectors have been identified as proteases or protease inhibitors which act to functionally defend or camouflage the pathogens from plant proteases and immune receptors. In this review, we discuss known protease functions and protease-regulated signaling processes involved in both sides of plant–pathogen interactions.

Introduction

Plants are sessile factories of biologically useful carbon. It is therefore not surprising that they are continuously assaulted by pests and pathogens which seek to assimilate plant-derived nutrients to survive and propagate. In response, plants have evolved a sophisticated immune system which is distinct from mammalian immunity in that it lacks adaptive structures (e.g. antibodies) and consists of no specialized immune cells [1]. Primarily, plants actuate immunity by strengthening existing physical and chemical bulwarks, and activating two types of innate immune signaling pathways: pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) [2,3]. Local induction of plant immunity also triggers plant systemic acquired resistance (SAR) to subsequent infections in distal tissues [1].

PTI is considered the first line of plant-inducible defense and is canonically triggered through the recognition of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) by plasma membrane-localized pattern recognition receptors (PRRs) [4,5]. ETI, in contrast, is a second layer of inducible defense typically activated by the intracellular recognition of pathogen effector molecules by plant resistance (R) gene products, the majority of which belonging to nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins [2,3]. ETI is often characterized by localized programmed cell death (PCD) — a process designated the ‘hypersensitive response’ (HR). Both PTI and ETI consist of perception, signal transduction, and finally, the immune response output. Each of these steps is extensively regulated by plants to maximize defense without detrimental effect and is conversely targeted by pathogens to promote infections for the benefit of pathogen growth (Figures 1 and 2).

Schematic of plant protease-regulated immune signaling pathways.

Figure 1.
Schematic of plant protease-regulated immune signaling pathways.

Plant proteases regulate multiple immune signaling pathways, including apoplastic immunity, PTI, and ETI. In plant apoplast, some peptides, such as systemin, Pep1, PIP1, GmSubPep, RALF23, GRI, and KOD, can regulate plant PTI or activate PCD responses. The maturation processes of these peptides require plant subtilases, MCs, and some other proteases. Plant subtilases, such as S1P/SBT6.1 and SBT3.5, can regulate cell wall-mediated immunity by processing cell wall-modifying enzyme PMEs. In addition, some plant proteases also regulate plant PTI by cleaving PRRs, including CERK1 and Xa21. In oat chloroplasts, two subtilases, SAS-1 and SAS-2, are required for the cleavage of Rubisco protein to regulate victorin-induced cell death. In Arabidopsis, several MCs (AtMC1, AtMC2, and AtMC4) and the proteasome β1 subunit PBA1 differentially contribute to R protein and fungal toxin-triggered PCD. In vacuole, proteases, such as RD21, usually are processed by VPEs and regulate viral, fungal toxin, elicitor, and Avr effector-triggered PCD. The Arabidopsis serine protease inhibitor AtSerpin1, which localizes in the cytoplasm, interacts and inhibits the RD21 protease activity once the protease is released into the cytoplasm, to control host cell pro-death functions of RD21. The Arabidopsis subtilase SBT5.2 interacts with and prevents MYB30 from entering the nucleus, resulting in the suppresion of MYB30-regulated immune-related gene transcription.

Figure 1.
Schematic of plant protease-regulated immune signaling pathways.

Plant proteases regulate multiple immune signaling pathways, including apoplastic immunity, PTI, and ETI. In plant apoplast, some peptides, such as systemin, Pep1, PIP1, GmSubPep, RALF23, GRI, and KOD, can regulate plant PTI or activate PCD responses. The maturation processes of these peptides require plant subtilases, MCs, and some other proteases. Plant subtilases, such as S1P/SBT6.1 and SBT3.5, can regulate cell wall-mediated immunity by processing cell wall-modifying enzyme PMEs. In addition, some plant proteases also regulate plant PTI by cleaving PRRs, including CERK1 and Xa21. In oat chloroplasts, two subtilases, SAS-1 and SAS-2, are required for the cleavage of Rubisco protein to regulate victorin-induced cell death. In Arabidopsis, several MCs (AtMC1, AtMC2, and AtMC4) and the proteasome β1 subunit PBA1 differentially contribute to R protein and fungal toxin-triggered PCD. In vacuole, proteases, such as RD21, usually are processed by VPEs and regulate viral, fungal toxin, elicitor, and Avr effector-triggered PCD. The Arabidopsis serine protease inhibitor AtSerpin1, which localizes in the cytoplasm, interacts and inhibits the RD21 protease activity once the protease is released into the cytoplasm, to control host cell pro-death functions of RD21. The Arabidopsis subtilase SBT5.2 interacts with and prevents MYB30 from entering the nucleus, resulting in the suppresion of MYB30-regulated immune-related gene transcription.

Schematic of mechanisms of pathogen effectors with protease or protease inhibitor activities.

Figure 2.
Schematic of mechanisms of pathogen effectors with protease or protease inhibitor activities.

Plant pathogens promote infections through the secretion of multiple effector proteins into plant apoplast, cytosol, or nucleus. Some of effectors possess protease activities. For example, Pst DC3000 secretes a metalloprotease, AprA, which can cleave flagellin monomers, to prevent flagellin from recognition by FLS2. Some fungi secrete effector proteases, such as fungalysin, FoSep1, and FoMep1, that cleave plant chitinases and β-1,3-glucanases, to suppress plant apoplastic immunity. Proteases in the gut of herbivorous insects are able to cleave plant chloroplastic ATP synthase and release Inceptin to trigger immune and wounding responses in cowpea. To counteract the activity of plant proteases, some fungi also secrete effectors that inhibit the activities of plant proteases, such as Avr2 from C. fulvum, EPIs from P. infestans, and Pit2 from U. maydis. In plant cytoplasm, bacterial T3E proteases target and cleave different plant immune signaling components to suppress plant immunity. For example, P. syringae effector HopB1 and HopAR1 target and cleave PTI mediator BAK1 and BIK1, respectively, to block PTI. P. syringae effector AvrRpt2 and HopX1 cleave and subsequently stimulate the ubiquitin/proteasome-dependent degradation of Aux/IAA and JAZ proteins to promote auxin and JA signaling, respectively. HopZ1 interacts with and degrades the isoflavone biosynthesis enzyme GmHID1 to suppress isoflavone biosynthesis. XopJ and HopZ4 effectors degrade the proteasome subunit RPT6 to suppress proteasome activity and abolish SA-dependent immune responses. P. syringae effector HopN1 targets and degrades photosystem II component PsbQ to inhibit pathogen-induced PCD. In nucleus, Xcv effector XopD destabilizes the ethylene responsive transcription factor SlERF4 by cleaving SIERF4-associated SUMO isoforms and thus represses ethylene-induced gene transcription. To antagonize the virulence caused by pathogen effectors, plants have evolved ETI. For instance, the suppression of the protease activity of tomato RCR3 by C. fulvum effector Avr2 triggers Cf-2-mediated ETI. Rice R protein Pi-ta activates ETI through direct recognition of M. grisea effector Avr-Pita. Arabidopsis RPS5 and RPS2 trigger ETI by perceiving the P. syringae effector AvrPphB and AvrRpt2-induced cleavage and degradation of host proteins PBS1 and RIN4, respectively. Arabidopsis R protein RRS1-R activates ETI upon R. solanacearum PopP2 effector targeting host RD19 protease.

Figure 2.
Schematic of mechanisms of pathogen effectors with protease or protease inhibitor activities.

Plant pathogens promote infections through the secretion of multiple effector proteins into plant apoplast, cytosol, or nucleus. Some of effectors possess protease activities. For example, Pst DC3000 secretes a metalloprotease, AprA, which can cleave flagellin monomers, to prevent flagellin from recognition by FLS2. Some fungi secrete effector proteases, such as fungalysin, FoSep1, and FoMep1, that cleave plant chitinases and β-1,3-glucanases, to suppress plant apoplastic immunity. Proteases in the gut of herbivorous insects are able to cleave plant chloroplastic ATP synthase and release Inceptin to trigger immune and wounding responses in cowpea. To counteract the activity of plant proteases, some fungi also secrete effectors that inhibit the activities of plant proteases, such as Avr2 from C. fulvum, EPIs from P. infestans, and Pit2 from U. maydis. In plant cytoplasm, bacterial T3E proteases target and cleave different plant immune signaling components to suppress plant immunity. For example, P. syringae effector HopB1 and HopAR1 target and cleave PTI mediator BAK1 and BIK1, respectively, to block PTI. P. syringae effector AvrRpt2 and HopX1 cleave and subsequently stimulate the ubiquitin/proteasome-dependent degradation of Aux/IAA and JAZ proteins to promote auxin and JA signaling, respectively. HopZ1 interacts with and degrades the isoflavone biosynthesis enzyme GmHID1 to suppress isoflavone biosynthesis. XopJ and HopZ4 effectors degrade the proteasome subunit RPT6 to suppress proteasome activity and abolish SA-dependent immune responses. P. syringae effector HopN1 targets and degrades photosystem II component PsbQ to inhibit pathogen-induced PCD. In nucleus, Xcv effector XopD destabilizes the ethylene responsive transcription factor SlERF4 by cleaving SIERF4-associated SUMO isoforms and thus represses ethylene-induced gene transcription. To antagonize the virulence caused by pathogen effectors, plants have evolved ETI. For instance, the suppression of the protease activity of tomato RCR3 by C. fulvum effector Avr2 triggers Cf-2-mediated ETI. Rice R protein Pi-ta activates ETI through direct recognition of M. grisea effector Avr-Pita. Arabidopsis RPS5 and RPS2 trigger ETI by perceiving the P. syringae effector AvrPphB and AvrRpt2-induced cleavage and degradation of host proteins PBS1 and RIN4, respectively. Arabidopsis R protein RRS1-R activates ETI upon R. solanacearum PopP2 effector targeting host RD19 protease.

One way for pathogens to interact with plants is to secret effector proteins into the apoplastic space and plant cytoplasm [6]. An important subset of these effectors are known to be proteases or protease inhibitors, and accumulating evidence suggests that they may function to dismantle host perception machinery, degrade host defense structures, or degrade/inhibit host-derived proteases (Figure 2) [6,7]. Therefore, if the plant–pathogen dialog is viewed through the colorful metaphor of warfare, proteases can be thought of as a tool for avoiding detection (the cloak), physically damaging one's opponent (the dagger), and defending from such an attack (the shield).

In this review, we provide an overview of protease functions and protease-regulated signaling processes involved in both plant immunity and pathogen pathogenicity. Despite that ubiquitin-directed proteasome activity is critically important in plant immune function, that subset of proteolysis will not be discussed in details in this review with the brief exception of the Arabidopsis proteasome β1 subunit PBA1. This review is complementary to two recent reviews about the role of proteases in plant immunity [8,9].

A summary of plant-derived proteases

The term ‘protease’ (also called peptidases or proteinases) refers to any enzyme that performs protein catabolism by hydrolysis of peptide bonds (proteolysis). In plants, proteases represent a superfamily of proteins which compose ∼2% of coding genes. Arabidopsis and rice, respectively, encode over 800 and 600 proteases which are distributed about 60 families and 30 clans predicted through sequence similarity (MEROPS peptidase database, http://merops.sanger.ac.uk/) [10]. Based on their proteolytic mechanisms, plant proteases are divided into four groups: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. Cysteine and serine proteases are named for the amino acid residue in their active site which is used as a nucleophile for the formation of an acyl intermediate during proteolytic cleavage [10]. Aspartic proteases, however, rely on a distinct catalytic mechanism which employs two aspartic acid residues located within the conserved Asp-Thr/Ser-Gly motifs. Metalloproteases, in contrast, make use of a metal cofactor in their catalytic activity and, like aspartic proteases, use water as their nucleophile [10].

Plant cysteine proteases comprise 15 families grouped into five clans. Most plant cysteine proteases belong to the C1 family proteases, which were also named papain-like cysteine proteases (PLCPs) [11]. Plant serine proteases contain 14 families divided into nine clans. Plant subtilisin-like proteases (subtilases) corresponding to the S8A subfamily of serine proteases represent the most extensively studied group of proteases [11]. Plant aspartic proteases are distributed to five families and two clans, but the majority belongs to the A1 family proteases, which were termed pepsin-like aspartic proteases (PLAPs) [11]. Finally, plant metalloproteases are grouped into 19 families and over 11 clans [11]. Currently, ∼30 plant proteases, covering all four catalytic classes, have been identified to be involved in plant resistance against various pathogens in model plants and crops in the past several decades (Table 1). Some plant proteases are localized to the apoplast where they modify cell surface receptor configurations and extracellular matrix proteins; whereas others are localized to the cytoplasm, vacuole, or nucleus, where they cleave plant immune components to modulate plant immune signaling or to activate PCD (Figure 1).

Table 1
Plant proteases with known functions in plant immunity
Protease name Species Family Subcellular localization Cleavage target Function Reference 
RCR3 Tomato PLCP Apoplast Unknown Resistance to C. fulvum, P. infestans, and G. rostochiensis; Avr2-triggerred HR [6166,112
Pip1 Tomato PLCP Apoplast Unknown Resistance to P. infestans [14,15
C14 Tomato PLCP Apoplast Unknown Resistance to P. infestans [16
XCP2 Arabidopsis PLCP Apoplast Unknown Resistance to R. solanacearum [17
CYP1, CYP2 Tomato PLCP Apoplast Unknown Resistance to C. destructivum [18
P69B Tomato Serine protease Apoplast Unknown Apoplastic immunity [1921,92
P69C Tomato Serine protease Apoplast LRP Apoplastic immunity [1922
SBT3.3 Arabidopsis Serine protease Apoplast Unknown Immune priming and disease resistance [23
SBT6.1/S1P Arabidopsis Serine protease Apoplast RALF23
PME1 
PTI
Apoplastic immunity 
[26,46,47
SBT3.5 Arabidopsis Serine protease Apoplast PME17 Apoplastic immunity [27
SlPhyt-1, SlPhyt-2 Tomato Serine protease Apoplast Prosystemin Systemin mature [35
Lap-A Tomato Aminopeptidase Apoplast l-systemin Systemin mature [36,37
SBT5.2b Arabidopsis Serine protease Endosome MYB30 Suppress MYB30-mediated resistance [58
RD19 Arabidopsis Cysteine protease Nucleus Unknown RRS1-R-mediated ETI [67
AtMC1 Arabidopsis Metalloprotease Apoplast Unknown PCD [75
AtMC2 Arabidopsis Metalloprotease Apoplast Unknown PCD [75
AtMC4 Arabidopsis metalloprotease Cytosol Unknown PCD [76
AtMC9 Arabidopsis Metalloprotease Cytosol Unknown PCD [80
VPEs Arabidopsis, tomato, and tobacco Cysteine protease Vacuole Unknown PCD [8183,85
RD21 Arabidopsis PLCP Vacuole  OA-triggered PCD [77
CathB Tobacco PLCP Apoplast Unknown HR [88
CEP1 Arabidopsis PLCP Endoplasmic reticulum Unknown PCD and disease resistance [89,90
PBA1 Plants DEVDase Cytosol and nucleus Ubiquitin-tagged proteins Bacteria-induced HR [91
Phytaspase Tobacco Serine protease Apoplast Unknown PCD [94
Saspases Oat Serine protease Apoplast Rubisco protein Victorin-induced PCD [96
APCB1 Arabidopsis Aspartic protease Apoplast BAG6 Autophagy and disease resistance [99
CDR1 Arabidopsis Aspartic protease Apoplast Unknown SAR and PCD [101
AED1 Arabidopsis Aspartic protease Apoplast Unknown Autophagy [102
Protease name Species Family Subcellular localization Cleavage target Function Reference 
RCR3 Tomato PLCP Apoplast Unknown Resistance to C. fulvum, P. infestans, and G. rostochiensis; Avr2-triggerred HR [6166,112
Pip1 Tomato PLCP Apoplast Unknown Resistance to P. infestans [14,15
C14 Tomato PLCP Apoplast Unknown Resistance to P. infestans [16
XCP2 Arabidopsis PLCP Apoplast Unknown Resistance to R. solanacearum [17
CYP1, CYP2 Tomato PLCP Apoplast Unknown Resistance to C. destructivum [18
P69B Tomato Serine protease Apoplast Unknown Apoplastic immunity [1921,92
P69C Tomato Serine protease Apoplast LRP Apoplastic immunity [1922
SBT3.3 Arabidopsis Serine protease Apoplast Unknown Immune priming and disease resistance [23
SBT6.1/S1P Arabidopsis Serine protease Apoplast RALF23
PME1 
PTI
Apoplastic immunity 
[26,46,47
SBT3.5 Arabidopsis Serine protease Apoplast PME17 Apoplastic immunity [27
SlPhyt-1, SlPhyt-2 Tomato Serine protease Apoplast Prosystemin Systemin mature [35
Lap-A Tomato Aminopeptidase Apoplast l-systemin Systemin mature [36,37
SBT5.2b Arabidopsis Serine protease Endosome MYB30 Suppress MYB30-mediated resistance [58
RD19 Arabidopsis Cysteine protease Nucleus Unknown RRS1-R-mediated ETI [67
AtMC1 Arabidopsis Metalloprotease Apoplast Unknown PCD [75
AtMC2 Arabidopsis Metalloprotease Apoplast Unknown PCD [75
AtMC4 Arabidopsis metalloprotease Cytosol Unknown PCD [76
AtMC9 Arabidopsis Metalloprotease Cytosol Unknown PCD [80
VPEs Arabidopsis, tomato, and tobacco Cysteine protease Vacuole Unknown PCD [8183,85
RD21 Arabidopsis PLCP Vacuole  OA-triggered PCD [77
CathB Tobacco PLCP Apoplast Unknown HR [88
CEP1 Arabidopsis PLCP Endoplasmic reticulum Unknown PCD and disease resistance [89,90
PBA1 Plants DEVDase Cytosol and nucleus Ubiquitin-tagged proteins Bacteria-induced HR [91
Phytaspase Tobacco Serine protease Apoplast Unknown PCD [94
Saspases Oat Serine protease Apoplast Rubisco protein Victorin-induced PCD [96
APCB1 Arabidopsis Aspartic protease Apoplast BAG6 Autophagy and disease resistance [99
CDR1 Arabidopsis Aspartic protease Apoplast Unknown SAR and PCD [101
AED1 Arabidopsis Aspartic protease Apoplast Unknown Autophagy [102

Like all other eukaryotes, plants employ a pervasive proteolysis system known as the 26S proteasome. The 26S proteasome is a multisubunit protease complex responsible for degrading a wide range of intracellular proteins, which are usually targeted for this degradation through the covalent attachment of a small protein tag known as ubiquitin [12]. Plants also encode a group of deubiquitinating enzymes, ubiquitin-specific proteases (UBPs), which cleave ubiquitin from target proteins to inhibit ubiquitin-mediated protein degradation [13].

Plant-derived proteases in apoplast-mediated defense

The apoplast is an early battleground in plant–pathogen interactions where the plant constitutively and inducibly accumulates defense-related proteins including proteases. Some apoplastic PLCPs, such as tomato Phytophthora-inhibited protease 1 (Pip1) [14,15], tomato and tobacco C14 [16], Arabidopsis xylem cysteine protease 2 (XCP2) [17], and tomato Cys proteases 1 and 2 (CYP1 and CYP2) [18], have been implicated in plant resistance to various pathogens. Silencing of the PLCP C14 in Nicotiana benthamiana significantly increased susceptibility to Phytophthora infestans [16]. Loss-of-function mutations in the Arabidopsis PLCP XCP2 caused an increased susceptibility to Ralstonia solanacearum [17]. Similarly, silencing CYP1 or CYP2 in N. benthamiana increased its susceptibility to the necrotrophic fungal pathogen Colletotrichum destructivum [18]. However, the molecular actions, such as targets and specificity, underlying these protease-related resistances have yet to be elucidated.

Tomato P69, a subtilase (SBT) family serine protease, was originally characterized as a pathogenesis-related (PR) protein found in tomato extracellular fluid (PR-7) with a molecular mass of 69 kDa [19]. The tomato genome carries six P69 (P69A–P69F) genes [20]. Of these six, two (P69B and P69C) are known to be systemically induced in expression by both pathogen infection and exogenous treatment with salicylic acid (SA), a plant defense hormone [20,21]. P69C was found to cleave an extracellular matrix-associated leucine-rich repeat protein (LRP) of unknown function in tomato [22]. AtSBT3.3, an ortholog of tomato P69C in Arabidopsis, positively regulates plant resistance to bacterium Pseudomonas syringae pv. tomato (Pst) DC3000 and obligates oomycete Hyaloperonospora arabidopsidis [23]. The enhanced resistance in AtSBT3.3 overexpression plants is probably associated with SA-mediated priming of immune genes, the expression of which might be regulated by chromatin remodeling [23]. The substrate(s) of AtSBT3.3 remains unclear.

Plant cell walls act as barriers for physical defenses — a role particularly important in their defense against fungal necrotrophs [24]. Upon pathogen attacks, plants employ several defense mechanisms which rely on the biochemical modification of cell wall components. The pectin matrix is a major structural component of the cell wall in Arabidopsis. Demethylesterification of pectin is considered to be an important step for mechanical strength of cell walls [25]. In Arabidopsis, pectin methylesterases (PMEs) are responsible for demethylesterification of homogalacturonans (HGs), the major pectic constituent of cell walls. Certain evidence suggests that, in several cases, Arabidopsis PMEs can be cleaved by SBTs. It was demonstrated that the N-terminal propeptide region of AtPME1 can be proteolytically removed by SITE-1 PROTEASE (AtS1P)/SBT6.1 to regulate the release of mature PME1 from the Golgi to the extracellular space [26]. Additionally, AtSBT3.5 probably processes AtPME17 at a specific single processing motif to release the mature protein into the apoplast [27]. There are 66 PME genes in Arabidopsis. While there is no direct evidence showing that SBT-mediated cleavage of PMEs plays a role in plant resistance, it was reported that AtPME1, AtPME17, as well as other PMEs contribute to immunity against P. syringae [28]. Furthermore, three PME inhibitors can protect the cell wall integrity of Arabidopsis against the fungal necrotrophic pathogen Botrytis cinerea [29].

Plant-derived proteases in microbe-, herbivore- or danger-associated molecular pattern-activated immunity

Plant innate immune signaling is classically initiated with membrane-localized PRRs upon recognition of microbe-, herbivore- or danger-associated molecular pattern (MAMP/HAMP/DAMP) ligands [4,5]. Plant PRRs are often single transmembrane domain-containing protein serine/threonine/tyrosine kinases called ‘receptor-like kinases’ (RLKs) or their related kinase-deficient ‘receptor-like proteins’ (RLPs) [4,5,30]. Once activated, PRRs relay signaling to an array of intracellular events, e.g. activation of receptor-like cytoplasmic kinases (RLCKs), mitogen-activated protein kinase cascades, and calcium-dependent protein kinases, production of reactive oxygen species (ROS), and regulation of transcriptional, metabolomic, and phytohormone outputs [4,5]. Proteases play a non-trivial role in this complex signaling.

Proteases as generators of endogenous DAMPs

Some plant RLKs can detect host-derived endogenous molecules, which are known as DAMPs and usually produced during pathogen infections or wounding and [31]. Endogenous peptides generated from plant protease-mediated cleavage of precursor proteins represent an important category of DAMPs. Systemin is the first endogenous peptide elicitor isolated from tomato [32]. This peptide is perceived by its receptors, leucine-rich repeat RLK (LRR-RLK) systemin receptor 1 (SYR1) and SYR2, to regulate wound-induced systemic defense responses and enhance resistance of tomato against herbivorous insects [33]. Systemin is an 18-amino acid peptide cleaved from the C-terminal region of a 200-amino acid precursor protein called prosystemin (PS) [34]. A recent report indicated that the cleavage of PS in tomato at two systemin-flanking aspartic acid residues can be performed by two phytaspases (highly specific subtisilin-like proteases), SlPhytaspase-1 (SlPhyt-1) and SlPhyt-2. This cleavage resulted in the production of systemin with an extra leucine (Leu) residue at the amino terminus (Leu-systemin) [35]. Destruction of the cleavage sites in PS not only prevented PS processing in vitro, but also eradicated systemin-inducible responses in vivo, indicating that phytaspase-dependent PS processing plays a crucial role in systemin signaling. Although Leu-systemin is biologically active, it is markedly less active than systemin. It was reported that Leu aminopeptidase A (LAP-A) can perform an N-terminal truncation of Leu-systemin [36], and that LAP-A deficiency impairs late wound response in tomato [37]. Thus, it was suggested that phytaspase and Leu aminopeptidase act sequentially to accomplish systemin maturation from PS during wounding. It was also reported that a 50-kDa unknown subtilisin/Kex2p-like endoprotease could cleave mature 18-amino acid systemin into two peptide fragments [38,39].

ELICITOR PEPTIDE 1 (PEP1) and PAMP-INDUCED PEPTIDE 1 (PIP1) are two endogenous peptide elicitors identified in Arabidopsis. PEP1, a 23-aa peptide, was the first endogenous peptide elicitor isolated from Arabidopsis [40]. Upon recognition by its receptors LRR-RLK PEPR1 and PEPR2, PEP1 activates typical PTI responses and enhances Arabidopsis resistance against hemibiotrophic bacterial P. syringae and necrotrophic fungal B. cinerea [41]. PEP1 is derived from the C-terminus of a 92-aa PEP1 pro-protein. Similarly, PIP1 is an 11-aa peptide cleaved from the C-terminus of the PIP1 pro-protein [42]. PIP1 also appears to activate typical PTI responses and contributes to Arabidopsis resistance to P. syringae and fungal Fusarium oxysporum upon recognition by LRR-RLK RLK7. GST-fused PIP1 pro-protein can be cleaved by apoplastic extracts of Arabidopsis leaves, suggesting that an extracellular protease(s) mediate(s) PIP1 cleavage [42].

CAP-DERIVED PEPTIDE 1 (CAPE1) is a peptide derived from the C-terminal end of tomato PR-1b [43]. It was suggested that CAPE1 was a DAMP, as it was induced by wounding and activated defense responses. However, it is still unclear how tomato PR-1b is cleaved to produce CAPE1. An unknown apoplastic aspartyl protease could be responsible for the PR-1b processing, as apoplastic aspartyl proteases have been reported to degrade PR proteins in tobacco and tomato [44]. Soybean (Glycine max) SUBTILASE PEPTIDE (GmSUBPEP) is a 12-aa endogenous peptide elicitor isolated from soybean leaves [45]. Exogenous application of synthetic GmSUBPEP on soybean leaves induced the expression of defense-related genes. Interestingly, when GmSUBPEP was synthesized with an additional amino acid on either the N- or C-terminus, a significant decrease in immune stimulation activity was observed, suggesting that small deviations from this 12-aa sequence greatly compromise its function [45].

Recently, Arabidopsis S1P/SBT6.1 was demonstrated to negatively regulate responses to the fungal MAMP chitin and bacterial MAMPs: flagellin-derived epitope peptide flg22 and elongation factor-Tu-derived peptide elf18. Furthermore, expression of S1P/STB6.1 reduced plant resistance to the Pst DC3000 phytotoxin coronatine-deficient mutant (cor) strain [46]. Similarly, a RAPID ALKALINIZATION FACTOR (RALF) peptide, RALF23, which is processed by S1P/SBT6.1, negatively regulates MAMP responses and resistance to Pst DC3000 (cor) [46,47]. Importantly, the heightened elf18-induced ROS production in the s1p-6 mutant was suppressed by exogenous application with the RALF23 peptide. This points to a hypothesis that the negative regulatory function of S1P/SBT6.1 in plant immunity is a result of RALF23 cleavage from its pro-protein, PRORALF23 [46]. Interestingly, RALF homologs were also found in many fungal phytopathogens [48]. It is possibly that fungal pathogens use functional RALF homologs to suppress plant immunity by inhibiting the formation of active receptor complexes, thereby enhancing their infections. The observed inhibition of the PTI by RALF23 relies on the Arabidopsis malectin-like receptor kinase FERONIA (FER), which was suggested to act as a scaffold to promote the formation of active heteromeric PRR signaling complexes. In response, MAMP perception appears to promote the processing of PRORALF23 by S1P/SBT6.1 [46]. This implies a possible negative-feedback mechanism to attenuate FER scaffolding and therefore control the duration and intensity of FER-mediated PTI.

Proteases as mediators of PRR cleavage and signaling

The extracellular cleavage of host receptor proteins leading to the release of soluble ectodomains is widely reported in animal cells — a process termed ‘ectodomain shedding’ [49]. Several lines of evidence suggest that this process is potentially important in plant immunity. The lysin motif (LysM) RLK CERK1 is the PRR for the perception of the fungal cell wall polysaccharide chitin and the bacterial peptidoglycan [4,5,30]. It was reported that Arabidopsis CERK1 undergoes ectodomain shedding. An amino acid mutation (L124 to F) in the second lysin motif (LysM) of the CERK1 ectodomain prevents this extracellular shedding. In effect, this mutation causes an enhanced pathogen-induced SA production and deregulation of cell death [50]. Interestingly, the enhanced defense caused by this mutation does not require CERK1 kinase activity, and this mutation does not alter chitin signaling. The authors suggest that CERK1 may possess a chitin-independent function in plant defense [50]. The shed ectodomain may suppress this activity. The identification of protease that mediates CERK1 ectodomain shedding will elucidate the mechanism and function of this interesting phenomenon in plant immunity.

The rice PRR Xa21, an LRR-RLK, confers resistance to several strains of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight disease in rice [51]. Xa21-mediated resistance is probably activated by a tyrosine-sulfated protein called RaxX (required for activation of Xa21-mediated immunity X) derived from Xoo [52]. While Xa21 is a known transmembrane protein, it contains a nuclear localization sequence (NLS) between its transmembrane and intracellular juxtamembrane domain [53]. This raises an interesting hypothesis that the cytoplasmic domain of Xa21 might be cleaved and imported into the nucleus. Indeed, Xa21-GFP cleavage products were demonstrated to localize in the nucleus in transient expression assays [53]. Moreover, the Xa21 intracellular domain can interact with several proteins predicted to be nuclear localized, including the WRKY62 transcription factor [54]. Consequently, the intracellular domain of Xa21 was suggested to play a role in transcriptional regulation. However, mutating the predicted NLS in Xa21 does not appear to affect resistance to Xoo [55], raising a question about the biological significance of Xa21 nuclear location. It also remains unknown for the protease that cleaves Xa21.

The Arabidopsis subtilase gene SBT5.2 produces two distinct transcripts encoding a secreted subtilase (SBT5.2a) and an intracellular protein (SBT5.2b). SBT5.2a was reported to process multiple peptide precursor proteins to generate mature peptide ligands, including IDA controlling floral organ abscission and EPF2 controlling stomatal patterning [56,57]. SBT5.2b, in contrast, does not possess proteolytic activity and localizes to vesicles where it interacts with and prevents MYB30 from entering the nucleus [58]. MYB30 is a transcription factor that regulates defense and localized cell death through the transcriptional activation of genes related to lipid biosynthesis [59]. Therefore, nuclear exclusion of MYB30 by SBT5.2b results in a reduction in MYB30-mediated transcription, thereby nullifying its resistance-conferring effects.

Plant-derived proteases as regulators of ETI and PCD

Proteases in ETI activation

ETI can be activated by both RLK/RLP transmembrane receptors and a family of polymorphic NBS-LRR R proteins in the cytoplasm. When pathogen-derived effectors, some called avirulence factors, are recognized by the corresponding R proteins, HR is typically triggered [60]. Studies indicate that some plant proteases are involved in R protein–effector complexing and therefore required for ETI activation (Figure 2).

Tomato Required for Cladosporium resistance 3 (RCR3) is a secreted PLCP with demonstrated proteolytic activity [61]. RCR3 was originally identified as a regulator in tomato ETI mediated by recognition between receptor-like protein Cf-2 and Cladosporium fulvum effector protein Avr2 [62]. Interestingly, RCR3 cysteine protease activity can be inhibited by a C. fulvum effector protein, Avr2. It has been proposed that the Rcr3–Avr2 interaction is essential to trigger Cf-2-mediated immunity [63,64]. RCR3 cysteine protease activity can be inhibited by Avr2, which functions as a protease inhibitor [63], suggesting that RCR3 is a virulence target of Avr2. Consistent with the hypothesis that virulence targets are important components in plant defense, RCR3 also contributes to tomato resistance to the late blight pathogen P. infestans [65], and the potato cyst nematode Globodera rostochiensis [66].

Another host-derived protease which probably mediates ETI activation is the Arabidopsis cysteine protease RESPOSNSE TO DEHYDRATION 19 (RD19) [67]. RD19 was identified as an interacting partner of the R. solanacearum PopP2 effector — a YopJ-like effector. PopP2 elicits disease resistance against R. solanacearum in Arabidopsis upon recognition by its cognate R protein, RRS1-R. RD19 protease activity is required for RRS1-R-dependent immune activation. Interestingly, RD19 was found to be localized in the vacuolar-associated mobile compartments in the absence of the PopP2 effector; however, association of PopP2 relocalizes RD19 and PopP2 complexes in the nucleus, which is probably required for the activation of RRS1-R-mediated defense [67]. It is unclear whether RD19-mediated cleavage of a host protein is required for RRS1-R activation.

Proteases in PCD regulation

As mentioned above, effector recognition through R proteins in plants often leads to localized PCD [60]. Elicitors and toxins produced by pathogens sometimes also trigger PCD. It was observed that PCD in plants shares several morphological and biochemical features with apoptosis in animals [68]. It was therefore hypothesized that the cellular machinery mediating cell death might be conserved in both animal apoptosis and plant PCD. Caspases, a group of cysteine-dependent aspartyl proteases which play a critical role in animal apoptosis, were one of the primary candidate gene families during the elucidation of PCD in plants [69]. Although plants do not have close homologs of caspases, some reports indicate that plants may contain enzymes with caspase-like activity involved in PCD [70]. Plants do possess a family of structurally related cysteine proteases called metacaspases (MCs), which contain the caspase-specific histidine and cysteine catalytic residues, as well as a conserved caspase-like secondary structure [71]. However, MCs cleave synthetic peptide substrates at arginine and lysine residues. This specificity is distinct from animal caspases, which cleave at the C-terminal side of an aspartate [72]. Vacuolar processing protease (VPEs), subtilases, and aspartic proteases are currently known to cleave after aspartate and recognize similar cleavage sites as caspases (Figure 1). It is possible that plant caspase activity is mediated by enzymes that are structurally distinct from animal caspases.

Metacaspases in PCD regulation

Metacaspases (MCs) belong to the CD clan of cysteine proteases [73]. The Arabidopsis genome encodes nine MCs (AtMC1–9), which are classified into two types, type I (AtMC1–3) and type II (AtMC4–9), based on primary sequence similarities and predicted tertiary domain structures. Type I AtMCs contain an N-terminal pro-domain and a C-terminal caspase-like domain, whereas type II AtMCs lack the pro-domain [74]. Different MCs may have different functions in PCD regulation. AtMC1 is a positive regulator of NBS-LRR R protein RPM1-mediated HR, whereas AtMC2 antagonizes AtMC1-mediated HR [75]. Both AtMC1 and AtMC2 contain a zinc-finger motif with sequence similarity to the negative cell death regulator LESION SIMULATING DISEASE 1 (LSD1) in their N-terminal pro-domain. Additionally, both contain highly conserved p20- and p10-like regions that show structural similarity to caspases. Their catalytic sites are composed of two cysteine residues near the C- and N-termini of the p20-like domain. In addition, AtMC4 was reported to be required for P. syringae effector AvrRpt2-triggered HR in Arabidopsis [76]. The substrates of AtMC1, AtMC2, and AtMC4 remain to be discovered.

Plant pathogens can induce necrosis- or PCD-like cell death in infected tissues by releasing numerous toxins, such as fumonisin B1 (FB1), oxalic acid (OA), and victorin [7779]. Studies indicate that plant proteases play important roles in toxin-induced cell death. FB1 is one kind of sphinganine analog mycotoxins produced by necrotrophic fungal Alternaria and Fusarium spp. FB1 elicits an apoptotic form of PCD in plants [79]. It has been reported that the type II MC AtMC4 is a positive regulator of FB1-induced cell death [76]. Arabidopsis mc4 mutant leaves exhibited reduced cell death in response to FB1, whereas overexpression of AtMC4 enhanced FB1-induced cell death. Furthermore, a mutation at the active cysteine of AtMC4 (C139A) eliminated the ability of this protease to trigger cell death. Thus, MCs can function in both effector-activated ETI and toxin-activated cell death.

It was shown that an 11-aa extracellular peptide, designated GRIp68–78, could be produced by AtMC9-mediated cleavage of the secreted protein GRIM REAPER (GRI) [80]. Once produced, this peptide binds the extracellular domain of the atypical LRR-containing RLK POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5). This interaction, in turn, induces SA and ROS-mediated cell death [80].

Other cysteine proteases in PCD regulation

Most mature plant cells contain vacuoles that accumulate proteins, including enzymes responsible for PCD. Vacuolar proteins are usually synthesized in the endoplasmic reticulum (ER) as larger precursors and then transported into vacuoles where these precursors are processed into their mature forms by VPEs. VPEs are typical cysteine proteases which act on similar cleavage sites to caspases, and are inhibited by caspase inhibitors [81]. Therefore, it was suggested that VPEs are the plant functional orthologs of animal caspases. Studies indicate that VPEs are involved in pathogen effector-, toxin-, and elicitor-induced PCD [8183]. For example, tobacco mosaic virus (TMV) causes the HR response in tobacco plants that carry the TMV-specific NBS-LRR protein N. Silencing of VPEs in N. benthamiana abolishes N-mediated PCD [82]. It was further demonstrated that tobacco VPE has caspase-1-like (YVADase) activity, which mediates a process of vacuolar collapse, liberating vacuolar hydrolytic enzymes into the cytoplasm to prevent viral proliferation [82].

The above-mentioned FB1-induced cell death was also accompanied by tonoplast disintegration and vacuolar collapse followed by lesion formation [84]. Four VPE-encoding genes have been identified in Arabidopsis: αVPE, βVPE, γVPE, and δVPE. The FB1-induced cell death was completely eliminated in the Arabidopsis quadruple vpe-null mutant. Of all four VPEs, γVPE is the most essential for FB1-induced cell death in Arabidopsis leaves. Overexpression of Arabidopsis vacuolar γVPE enhanced ion leakage, and γvpe single mutants showed less FB1-induced cell death [82,85].

Necrotrophic fungal pathogens, such as B. cinerea and Sclerotinia sclerotiorum, kill host cells through the secretion of considerable amounts of toxin OA. OA activates host anion channels and destabilizes vacuolar integrity [86]. The vacuolar protease RESPONSIVE TO DESICCATION 21 (RD21) was identified as a pro-death signal activated during the process of OA-stimulated cell death. RD21 is a PLCP [77]. It is present in both the ER and the vacuole itself [87]. Arabidopsis rd21 knockout mutants do not exhibit necrotroph- and OA-triggered PCD and are therefore more susceptible to these pathogens. Interestingly, the Arabidopsis serine protease inhibitor AtSERPIN1, which localizes to the cytoplasm, can interact with and inhibit RD21 protease activity, thus controlling host cell pro-death functions of the protease RD21 [77].

Tobacco papain cysteine protease cathepsin B NbCATHB is required for PCD in different pathosystems, including bacterial Erwinia amylovora-mediated non-host HR in tobacco and P. infestans Avr3a and potato R3a interaction-triggered HR [88]. Significantly, NbCATHB is not required for C. fulvum Avr4 and tomato Cf-4 recognition-mediated HR, suggesting the specificity of cathepsin B in cell death control. In addition, plants also possess a specific group of papain-like cysteine endopeptidases (CEPs), which are characterized by a C-terminal KDEL ER retention signal [89]. Arabidopsis has three CEPs: AtCEP1, AtCEP2, and AtCEP3. AtCEP1 is specifically expressed in ER and accumulates around fungal haustoria upon powdery mildew Erysiphe cruciferarum infection. The cep1 knockout mutant accumulates less dead cells and shows enhanced susceptibility to this biotrophic pathogen compared with wild-type plants [89,90]. However, the biochemical mechanism used by NbCathB and CEP1 to mediate PCD is unknown.

Proteasome β1 subunit PBA in PCD regulation

Tonoplast disintegration is not always required for vacuole-mediated cell death. A second mechanism involving the fusion of the vacuolar membrane with the plasma membrane was also reported [91]. This membrane fusion discharges vacuolar hydrolytic enzymes into the extracellular matrix to defend against bacterial attack. Interestingly, the proteasome is required for this membrane fusion associated with Pst DC3000 avrRpm1-induced disease resistance and HR. The Arabidopsis proteasome has three catalytic subunits, β1 subunit PBA1, β2 subunit PBB, and β5 subunit PBE. It was shown that PBA1 carries plant caspase-3-like (DEVDase) activity. Consistently, Pst DC3000 avrRpm1-induced HR can be effectively blocked by a caspase-3 inhibitor. Plants with a transcriptionally silenced PBA1 had reduced DEVDase activity and lost membrane fusion upon Pst DC3000 avrRpm1 infection [91].

Subtilases in PCD regulation

P69B is a pathogen-responsive subtilisin-like protease. A high accumulation of the protease can induce cell death in tomato. It was shown that P69B-induced cell death is negatively regulated by two matrix metalloproteinases, tomato homologs of mammalian matrix metalloproteinases (SlMMPs), Sl2-MMP and Sl3-MMP. Silencing the two protease genes in tomato plants greatly increases P69B protein stability and induced P69B-dependent spontaneous cell death. P69B was indicated to be a substrate of Sl2-MMP and Sl3-MMP in vitro. Therefore, Sl2- and Sl3-MMP act upstream of P69B to inactivate P69B and negatively regulate cell death [92].

Human caspase-3 is capable of inducing cleavage of the VirD2 protein of Agrobacterium tumefaciens [93]. This proteolytic activity was also detected in vivo using a VirD2-based reporter protein in tobacco (Nicotiana tabacum) leaves quickly following induction of N protein-mediated PCD. A four-amino acid motif ‘VEID’ was found to be the optimal recognition motif for this cleavage. The protease was thus named ‘phytaspase’ (plant aspartate-specific protease) [94]. Mass spectrometric characterization of tobacco and rice phytaspases revealed that these proteases belong to the family of subtilisin-like serine proteases. Interestingly, phytaspase was transported from the apoplast into the cytoplasm once TMV-induced PCD occurs. It was hypothesized that plants exploit a clever strategy to control the localization of death protease(s) which are excluded (secreted) from healthy cells and allowed to enter the cell interior upon induction of PCD [94].

Victorin, an unusual cyclic pentapeptide, is a host selective toxin produced by the fungus Cochliobolus victoriae, and results in Victoria blight disease in oats. Victorin causes cell death and is required for C. victoriae pathogenicity on oats carrying the Vb gene [78]. In Arabidopsis thaliana, victorin also can induce PCD, and the sensitivity to victorin is associated with an NBS-LRR protein LOCUS ORCHESTRATING VICTORIN EFFECTS1 (LOV1), indicating that this PCD is probably related to R gene-mediated HR response [95]. Notably, victorin-induced cell death in oats involves the proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) at its N-terminus following lysine 14 [78]. The proteolysis of Rubisco was shown to be prevented by caspase-specific inhibitors, indicating the requirement of a caspase-like protease(s) for the cleavage. Two subtilases termed Saspase-1 (SAS-1) and SAS-2 were identified to be necessary for this cleavage, but both SAS-1 and SAS-2 cannot directly cleave the purified Rubisco [96]. It was then suggested that saspases are components of a PCD-induced protease cascade that ultimately leads to the activation of a protease that is targeted to the chloroplast to cleave Rubisco.

Aspartyl proteases in PCD regulation

Bcl-2-associated athanogene (BAG) proteins are an evolutionarily conserved family of co-chaperone modulators that regulate diverse physiological processes, including cell death, in mammals and plants [97]. This family of proteins were initially identified as interacting proteins of Bcl-2, a critical regulator of apoptosis in animals, and characterized by a BAG domain in their C-terminus. The BAG proteins interact with chaperone protein heat shock protein 70 (HSP70) through their BAG domains and maintain protein homeostasis by acting on HSP70 [68]. Arabidopsis contains seven BAG homologs (AtBAG1–7). AtBAG6 is required for basal immunity against B. cinerea. Overexpression of AtBAG6 induced cell death [98]. Cleavage of AtBAG6 was found in Arabidopsis after both B. cinerea infection and chitin treatment. AtBAG6 cleavage contributes to PCD and plant defense responses to B. cinerea. AtBAG6 contains a potential caspase-1 cleavage site (LATD motif) following its BAG domain. AtBAG6 cleavage is inhibited by a glutamate-to-alanine mutation in this LATD motif. An aspartyl protease (APCB1) and a C2 GRAM domain protein (BAGP1) are also required for BAG6 processing [99].

Unknown proteases in PCD regulation

A 25-amino acid peptide in the cytosol named kiss of death (KOD) can activate a PCD in Arabidopsis [100]. KOD transcripts were induced in leaves 4 h after infiltration with avirulent strain Pst DC3000 avrRpm1, but not with the virulent strain, suggesting that KOD-triggered PCD is related to ETI. KOD overexpression in transient assays and in stably transformed Arabidopsis lines resulted in an increase in VADase and DEVDase caspase-like activities, respectively. Caspase inhibitor p35 was able to block the effect of KOD overexpression, suggesting that an unknown protease(s) with caspase-like activity is involved in KOD-induced PCD [100].

Plant-derived proteases as regulators of systemic acquired resistance

SAR is characterized by the systemic induction of defense in the distal leaves after a local pathogen infection [1]. An extracellular aspartic protease, CONSTITUTIVE DISEASE RESISTANCE 1 (CDR1), was implicated in SAR [101]. Overexpression of CDR1 leads to constitutive activation of SA-dependent local defense responses and enhanced resistance to Pst DC3000 or P. syringae pv. maculicola. In addition, it contributes to the induction of defense responses in systemic leaves. It was suggested that CDR1 might process an apoplastic protein to release an endogenous peptide elicitor between 3 and 10 kDa in size that activates SA-dependent immune responses and enhances resistance in local and distal tissues [101].

Arabidopsis ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) is required for both SAR signal production in primarily infected leaves and SAR signal perception in distal tissues. Comparative proteomic analysis of apoplastic extracts from both avrRpm1-expressing wild-type and eds1 mutant plants led to the identification of an apoplastic aspartyl protease, APOPLASTIC, EDS1-DEPENDENT 1 (AED1) [102]. AED1 is ∼30% identical with CDR1 at the amino acid level. It appears that AED1 specifically regulates SAR, but not local defense. It was proposed that AED1 acts as a homeostatic regulator to limit SAR signaling and reallocate resources from defense to plant growth by cleaving apoplastic proteins [102].

Pathogen/herbivore-derived proteases as activators of plant immunity

Pathogen-derived proteases as MAMPs

Somewhat paradoxically, pathogen-derived proteases can act as triggers of plant immunity. The protease itself or its cleavage product may function as an MAMP. For example, a serine protease encoded by PrpL in Pseudomonas aeruginosa, as well as its close homolog ArgC in Xanthomonas campestris, triggers classical PTI responses in Arabidopsis, including MPK3/MPK6 activation, oxidative burst elicitation, and resistance to Pst DC3000 infection [103]. ArgC or PrpL protease variants without detectable proteolytic activity were greatly impaired in PTI response elicitation, indicating that, in this case, the PTI activation of these proteases is dependent on their catalytic activity. AsES (Acremonium strictum Elicitor and Subtilisin), a subtilisin-like protease produced by the strawberry fungal pathogen A. strictum, also was reported to activate plant immune responses. With a foliar spray, this protein conferred partial resistance in strawberry against Colletotrichum acutatum, the causal agent of strawberry anthracnose disease [104].

Herbivore-midgut proteases as generators of HAMPs

When cowpea (Vigna unguiculata) leaves were consumed by Spodoptera larvae, the cowpea chloroplastic ATP synthase γ-subunit regulatory region (cATPC) was found to be cleaved by pest-derived proteases. This proteolysis probably produces the cATPC cleavage product inceptin and several inceptin-related peptides in the insect gut [105,106]. Inceptin and its related proteins were demonstrated to increase production of jasmonic acid (JA), ethylene (ET), and SA. Because armyworm larvae assimilate the nitrogen in plant tissues through the proteolytic action of some proteases, such as trypsin and pepsin, it was predicted that a combination of these midgut proteases is responsible for the proteolysis of cATPC and the production of these peptide elicitors.

Pathogen-derived proteases as triggers of ETI

Some effectors that trigger ETI are functional proteases. In most cases, their protease activity is typically required to activate ETI through their cognate R protein. For example, rice plants carrying NBS-class R protein Pi-ta are resistant to Magnaporthe grisea strains that carry the effector Avr-Pita [107]. Pi-ta recognizes Avr-Pita through direct physical interaction. Avr-Pita is predicted to encode a protein with sequence similarity to fungal zinc metalloproteases [108]. A point mutation (Glu to Asp) in the protease consensus site of Avr-Pita eliminated its ability to trigger Pi-ta-mediated resistance [108]. It is likely that the Pi-ta protein is a substrate of Avr-Pita protease. Cleavage of Pi-ta by AvrPi-ta could eliminate a domain of Pi-ta for self-inhibition and lead to its activation. Alternatively, Avr-Pita could activate Pi-ta by cleavage of another component in the complex.

Plant R proteins often do not directly recognize pathogen effectors [2,3]. They are activated by interacting with or otherwise monitoring host effector targets which are enzymatically modified by pathogen-derived effectors. For example, P. syringae AvrRpt2 and AvrPphB are two well-studied effectors that encode functional proteases. These proteins cleave host target proteins and consequently activate ETI. The cleavage of Arabidopsis AvrPphB SUSCEPTIBLE 1 (PBS1) by the effector AvrPphB can activate the R protein RESISTANCE TO P. syringae 5 (RPS5) [109]. Similarly, the cysteine protease AvrRpt2 can cleave RPM1-INTERACTING PROTEIN 4 (RIN4), a negative regulator of the basal defense response, and cause RIN4 degradation, which is subsequently detected by the R protein RPS2 to activate ETI [110].

Pathogen-derived proteases as dampers of host immune signaling

Secretion of effector proteins is a common virulence strategy used by successful pathogens to disrupt host immune signaling pathways [6]. Pathogenic effector proteins can be generally divided into two groups: extracellular effectors and cytoplasmic effectors. Extracellular effectors are secreted into the apoplast or xylem of host plants, while cytoplasmic effectors are translocated into the cytoplasm of host cells by bacterial type III secretion systems (T3SSs) or fungal haustoria. Studies indicated that many pathogenic effectors are active proteases or plant protease inhibitors that suppress host immunity by cleaving plant immune signaling components, or by suppressing plant protease-involved immunity [7] (Figure 2).

Apoplastic effectors disable plant apoplastic immunity

Escaping MAMP recognition by plant PRRs is one of the strategies used by pathogen to subvert PTI responses. Epitopes of bacterial flagellin constitute one important group of MAMPs that are recognized by Toll-like receptor 5 (TLR5) in mammals and by FLAGELLIN SENSING 2 (FLS2) in plants [111]. It was reported that P. aeruginosa, an opportunistic pathogen in humans and plants, secretes an alkaline protease called AprA that degrades flagellin monomers to prevent flagellin recognition by TLR5 and FLS2 [112]. AprA is a zinc metalloprotease. It is present in many plant pathogenic bacteria, including Pst DC3000. The cleavage of flagellin monomers by AprA in Pst DC3000 is required for its full virulence in both Arabidopsis and tomato. Flagellin is the principal component of bacterial flagellum, which is essential for bacterial motility. Importantly, flagellin can only be degraded by AprA in its monomeric form, suggesting that pathogens specifically employ AprA to eliminate host immune activation without compromising the integrity of the entire flagellum [112,113].

Several pathogenic effectors have been identified to possess protease activity to degrade or modify host defenses in the apoplastic space [7]. Plant apoplastic defenses include production of PR proteins such as chitinases, β-1,3-glucanases, and antifungal proteases. In response, it appears that some fungal metalloproteases, such as F. oxysporum FoSep1 and FoMep1, can cleave host-derived CBD-chitinases and β-1,3-glucanases [114]. Similarly, Fusarium verticillioides was shown to secrete the metalloprotease fungalysin to degrade class IV chitinases [84].

Plant pathogens also secrete protease inhibitors (PIs) into the apoplastic space to inhibit plant extracellular proteases and overcome protease-mediated defense. Avr2, for example, is a small cysteine-rich effector protein secreted by C. fulvum which inhibits the tomato apoplastic PLCPs RCR3 and Pip1 to support growth of C. fulvum in the apoplast [115]. Plants expressing Avr2 also exhibited increased susceptibility to other pathogenic fungi, such as B. cinerea and Verticillium dahliae [115]. Two other PIs from the oomycete pathogen P. infestans are the cystatin-like extracellular proteinase inhibitor (EPI) EPIC1 and EPIC2B [65]. These PIs selectively target RCR3, Pip1, and C14 in the apoplast of potato and tomato. The plant-parasitic nematode G. rostochiensis secretes the VAP1 effector that perturbs RCR3's activity [66]. In addition, P. infestans secretes two serine PIs (EPI1 and EPI10) which target and inactivate the major apoplastic subtilase P69B [116,117]. Furthermore, the maize pathogen Ustilago maydis secretes the cysteine PI, Pit2, which strongly inhibits three defense-related maize cysteine proteases, Cys protease 2 (CP2), and its isoforms CP1A and CP1B [118,119].

Pathogen effector-mediated cleavage blocks PTI signaling

Bacterial type III effectors (T3Es) usually block immune responses by interacting with and modifying intracellular host targets [6,7]. Some T3Es exhibit protease activity, cleaving key immune signaling components involved in various plant immune signaling pathways. For example, BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) acts as an important coreceptor involved PTI activation upon recognition of multiple MAMPs by LRR-RLK-type PRRs. Upon ligand perception, BAK1 is quickly recruited into a receptor complex and subsequently initiates downstream immune signaling [120]. Multiple T3Es, including AvrPto, AvrPtoB, and HopF2, from Pst DC3000 can target the intracellular kinase domain of BAK1 to inhibit PTI signaling [121]. Another Pst DC3000 T3E HopB1 also targets BAK1. HopB1 is an unconventional serine protease which specifically cleaves BAK1 between its Arg297 and Gly298 residues in an flg22-inducible manner, therefore blocking BAK1-mediated PTI and enhancing Pst DC3000 virulence [122]. Another critical regulator of PTI signaling is the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1). Active PRR complexes activate BIK1 through phosphorylation, resulting in its dissociation from the receptor complex. Once liberated, BIK1 phosphorylates downstream components, such as NADPH oxidase RbohD, to regulate immune responses [123,124]. The Pst DC3000 effector HopAR1 is a papain superfamily cysteine protease which enhances the virulence of P. syringae by cleaving BIK1 and its homologous proteins to block PTI signaling [125].

Pathogen effector-mediated cleavage in phytohormone manipulation

Plant immunity relies on a complex network of plant hormone signaling pathways [126]. SA, JA, and ET are the well-studied plant hormones involved in plant immunity. Generally, SA signaling mediates resistance against biotrophic and hemibiotrophic microbes such as P. syringae, whereas a combination of the JA and ET pathways activates resistance against necrotrophs such as the fungal pathogen B. cinerea [127]. The SA and JA/ET defense pathways often antagonize each other. Other plant hormones, such as auxins, gibberellins, and brassinosteroids, have also been associated with plant immunity [128130]. Several lines of evidence also indicated that pathogens enhance host susceptibility by manipulating these hormone signaling pathways.

Auxin negatively regulates plant resistance against P. syringae [128]. Exogenous application of synthetic auxin enhanced plant susceptibility to P. syringae infections, whereas mutant plants impaired in auxin signaling exhibited enhanced resistance [131,132]. P. syringae can suppress plant immunity and enhance the pathogenicity using the T3E AvrRpt2, a cysteine protease, which manipulates plant auxin signaling by stimulating the ubiquitin/proteasome-dependent degradation of Aux/IAA proteins, the key negative regulators in auxin signaling [133]. P. syringae effector HopX1 also encodes a cysteine protease that interacts with and promotes the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins. JAZ proteins are repressors of JA signaling, and therefore their degradation activates the JA signaling pathway and indirectly suppresses the SA pathway [134].

XopD, a T3E from X. campestris pv. vesicatoria (Xcv), contains an active plant-specific cysteine protease domain in its C-terminus that cleaves tomato and Arabidopsis small ubiquitin-like modification (SUMO) isoforms after invariant C-terminal diglycine residues [135]. In tomato, XopD desumoylates the ET-responsive transcription factor SlERF4 at its lysine 53 site, causing SlERF4 destabilization and repressing ethylene-induced transcription, which is required for ET production and resistance against Xcv infection [136].

Pathogen effector proteases block other immune signaling

The Yersinia outer protein J (YopJ) family of T3Es are also cysteine proteases [137]. The Xcv T3Es AvrXv4 and XopJ, and the P. syringae T3Es HopZ1 and HopZ4 belong to YopJ family T3Es. Like XopD, Xcv effector AvrXv4 was predicted to exhibit SUMO isopeptidase activity as AvrXv4 expression in planta leads to a reduction in SUMO-modified proteins [138]. However, the host target of AvrXv4 is still unknown. HopZ1 has been shown to be an active cysteine protease. It suppresses isoflavone biosynthesis by physically interacting with and degrading the isoflavone biosynthesis enzyme, 2-hydroxyisoflavanone dehydratase (GmHID1), and promotes infection in soybean [139]. However, HopZ1 also possesses acetyltransferase activity. It is unclear whether the degradation of GmHID1 is a result of its acetylation by HopZ1. Both XopJ and HopZ4 were shown to interact with and degrade the proteasomal subunit Regulatory Particle AAA-ATPase6 (RPT6) to suppress proteasome activity, resulting in the inhibition of SA-related immune responses [140,141]. P. syringae effector HopN1 is a cysteine protease which targets and degrades photosystem II subunit Q (PsbQ), a member of the oxygen evolving complex of photosystem II to inhibit pathogen-induced PCD [142].

Conclusion

Immune responses are initiated by plants to defend against pathogens upon perception of PAMPs, DAMPs, or effectors via PRRs or R proteins. To counteract plant defense, pathogens secrete an array of virulence effectors into apoplastic spaces and plant cells. The number of proteases implicated in the regulation of plant–pathogen interactions continues to grow on both the plant and the pathogen sides. From the side of plants, proteases appear to play critical roles in regulating various immune responses. Some proteases were shown to regulate apoplast-mediated defense by cleaving cell wall-modifying enzymes or other unknown targets, some are responsible for the maturation of peptide-type DAMPs to activate PTI, some are required for the recognition of pathogen effectors and ETI activation, and some are involved in the regulation of PCD and SAR (Figure 1). From the side of pathogens, a great number of effector proteins function as functional proteases that are employed by pathogens to impede plant immune signaling pathways, including PTI, ETI, immune-related hormone-regulated immunity, and so on. In addition, some effectors represent plant protease inhibitors which bind and inhibit plant protease activity, and thus block the plant proteases-mediated immunity (Figure 2).

Although great progress has been achieved for the elucidation of protease functions in plant–pathogen interactions, many questions concerning the biochemical roles and regulatory mechanisms of proteases in plant–pathogen interactions remain unresolved. Some peptide elicitors, plant immune regulators, and receptors are known to be processed by proteases, but very often these proteases and their specific cleavage sites have yet to be elucidated. To figure out the cleavage sites will help to understand the work modes of proteases. Although proteases have a demonstrable role in plant PCD during pathogen infection, the mechanisms underlying this role are only beginning to be understood. As crucial regulators of plant immune signaling, proteases appear to be carefully and specifically expressed, localized, regulated, sequestered, and degraded under every conceivable condition. As tools for cellular and molecular imaging become more sophisticated, the question of how plants control the spatiotemporal regulation of their proteases becomes even more salient. Furthermore, proteases have mainly been implicated in plant–pathogen interactions through a phenotypic characterization of protease mutants. This approach is limited because many proteases are members of a gene family, and therefore act in a redundant or semi-redundant fashion. A recent report effectively overcomes this problem through an expression-based approach whereby a protease promoter controls the expression of a protease inhibitor [57]. New methodologies of gene knockout, such as CRISPR/Cas9-mediated gene editing [143] and cell-type-specific proteomics, combining biochemical, cytological, and omics analyses will prove critical for a more thorough elucidation of the roles of proteases in plant–pathogen interactions. Approaching the answers to these questions will contribute to the generation of novel resistant germplasm and innovative techniques for disease management in agricultural contexts.

Abbreviations

     
  • AED1

    APOPLASTIC, EDS1-DEPENDENT 1

  •  
  • AsES

    Acremonium strictum Elicitor and Subtilisin

  •  
  • BAG

    Bcl-2-associated athanogene

  •  
  • BAK1

    BRI1-ASSOCIATED RECEPTOR KINASE 1

  •  
  • BIK1

    BOTRYTIS-INDUCED KINASE 1

  •  
  • CAPE1

    CAP-DERIVED PEPTIDE 1

  •  
  • cATPC

    chloroplastic ATP synthase γ-subunit regulatory region

  •  
  • CDR1

    CONSTITUTIVE DISEASE RESISTANCE 1

  •  
  • CEPs

    cysteine endopeptidases

  •  
  • CP2

    Cys protease 2

  •  
  • EDS1

    ENHANCED DISEASE SUSCEPTIBILITY 1

  •  
  • EPI

    extracellular proteinase inhibitor

  •  
  • ER

    endoplasmic reticulum

  •  
  • ET

    ethylene

  •  
  • ETI

    effector-triggered immunity

  •  
  • FB1

    fumonisin B1

  •  
  • FER

    FERONIA

  •  
  • FLS2

    FLAGELLIN SENSING 2

  •  
  • GmSUBPEP

    Glycine max SUBTILASE PEPTIDE

  •  
  • GRI

    GRIM REAPER

  •  
  • HAMPs

    herbivore-associated molecular patterns

  •  
  • HR

    hypersensitive response

  •  
  • HSP70

    heat shock protein 70

  •  
  • JA

    jasmonic acid

  •  
  • JAZ

    JASMONATE ZIM-DOMAIN

  •  
  • KOD

    kiss of death

  •  
  • LAP-A

    Leu aminopeptidase A

  •  
  • LRP

    leucine-rich repeat protein

  •  
  • LRR-RLK

    leucine-rich repeat RLK

  •  
  • MAMPs

    microbe-associated molecular patterns

  •  
  • MCs

    metacaspases

  •  
  • NBS-LRR

    nucleotide-binding site leucine-rich repeat

  •  
  • NLS

    nuclear localization sequence

  •  
  • OA

    oxalic acid

  •  
  • PAMPs

    pathogen-associated molecular patterns

  •  
  • PCD

    programmed cell death

  •  
  • PEP1

    ELICITOR PEPTIDE 1

  •  
  • PIP1

    PAMP-INDUCED PEPTIDE 1

  •  
  • PIs

    protease inhibitors

  •  
  • PLCPs

    papain-like cysteine proteases

  •  
  • PMEs

    pectin methylesterases

  •  
  • PR

    pathogenesis-related

  •  
  • PRRs

    pattern recognition receptors

  •  
  • PS

    prosystemin

  •  
  • PsbQ

    Photosystem II subunit Q

  •  
  • PTI

    pattern-triggered immunity

  •  
  • RALF

    RAPID ALKALINIZATION FACTOR

  •  
  • RaxX

    Required for activation of Xa21-mediated immunity X

  •  
  • RCR3

    Required for Cladosporium resistance 3

  •  
  • RD19

    RESPOSNSE TO DEHYDRATION 19

  •  
  • RIN4

    RPM1-INTERACTING PROTEIN 4

  •  
  • RLCK

    receptor-like cytoplasmic kinase

  •  
  • RLKs

    receptor-like kinases

  •  
  • RLPs

    receptor-like proteins

  •  
  • ROS

    reactive oxygen species

  •  
  • RPT6

    Regulatory Particle AAA-ATPase6

  •  
  • SA

    salicylic acid

  •  
  • SAR

    systemic acquired resistance

  •  
  • SAS

    Saspase

  •  
  • SBT

    subtilase

  •  
  • SUMO

    small ubiquitin-like modification

  •  
  • SYR1

    systemin receptor 1

  •  
  • TLR5

    Toll-like receptor 5

  •  
  • TMV

    tobacco mosaic virus

  •  
  • UBPs

    ubiquitin-specific proteases

  •  
  • VPEs

    vacuolar processing protease

  •  
  • Xcv

    X. campestris pv. vesicatoria

  •  
  • YopJ

    Yersinia outer protein J

Funding

This work was supported by the National Natural Science Foundation of China [31500971] and the State Scholarship Fund of China [201609995010] to S.H., and National Science Foundation [IOS-1252539] to P.H.

Acknowledgments

We apologize to our colleagues whose work was not discussed here because of space limitations.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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