Apoplastic Cell Death-Inducing Proteins of Filamentous Plant Pathogens: Roles in Plant-Pathogen Interactions

Filamentous pathogens, such as phytopathogenic oomycetes and fungi, secrete a remarkable diversity of apoplastic effector proteins to facilitate infection, many of which are able to induce cell death in plants. Over the past decades, over 177 apoplastic cell death-inducing proteins (CDIPs) have been identified in filamentous oomycetes and fungi. An emerging number of studies have demonstrated the role of many apoplastic CDIPs as essential virulence factors. At the same time, apoplastic CDIPs have been documented to be recognized by plant cells as pathogen-associated molecular patterns (PAMPs). The recent findings of extracellular recognition of apoplastic CDIPs by plant leucine-rich repeat-receptor-like proteins (LRR-RLPs) have greatly advanced our understanding of how plants detect them and mount a defense response. This review summarizes the latest advances in identifying apoplastic CDIPs of plant pathogenic oomycetes and fungi, and our current understanding of the dual roles of apoplastic CDIPs in plant-filamentous pathogen interactions.


INTRODUCTION
Filamentous pathogens, such as oomycetes and fungi, are the causal agents of many of the world's most serious plant diseases, causing extensive annual yield losses of crops worldwide (Giraldo and Valent, 2013;Sánchez-Vallet et al., 2018). Pathogenic oomycetes and fungi secrete a complex repertoire of effector proteins to establish successful interactions with host plants. These oomyceteor fungi-secreted effector proteins may function in the apoplast as well as within plant cells to interfere with host defense by a variety of mechanisms (Dou and Zhou, 2012;Lo Presti and Kahmann, 2017).
Studies from diverse filamentous pathogens have shown that many oomycete or fungal effector proteins possess the ability to induce cell death in plants (Gijzen and Nürnberger, 2006), including avirulence (AVR) proteins which trigger hypersensitive response (HR) upon recognition by cognate resistance (R) proteins (Kamoun, 2006;Ellis et al., 2009), nuclear-localized Crinkling and Necrosis proteins (CRNs) and nucleo-cytoplasmic RxLR proteins with capacity to induce plant cell death (Torto et al., 2003;Amaro et al., 2017), and extracellular cell death-inducing proteins (CDIPs) that function in the plant apoplast (Guo et al., 2019). The role of AVR proteins, CRN and RxLR effectors involved in the interactions of plants with filamentous pathogens have been reviewed extensively (Ellis et al., 2009;Giraldo and Valent, 2013;Amaro et al., 2017;Lo Presti and Kahmann, 2017). Here we focus this review on apoplastic CDIPs of filamentous plant pathogens and their roles in plantpathogen interactions.
Because cell death plays an important role in the interactions of plants with pathogens, there has been a long-standing interest in the characterization of pathogenic molecules which are able to induce plant cell death (Gijzen and Nürnberger, 2006). Since the characterization of elicitins, a family of conserved small secreted proteins from oomycetes that induce necrosis in Nicotiana species, in the 1980s (Billard et al., 1988;Huet and Pernollet, 1989;Ricci et al., 1989), a large number of apoplastic CDIPs have been identified in oomycete and fungal plant pathogens (Tables 1, 2). These apoplastic CDIPs induce plant cell death in a non-raceor non-species-specific manner, and were initially defined or considered as "elicitors" or "toxins" (Gijzen and Nürnberger, 2006;Derevnina et al., 2016). The role of apoplastic CDIPs in the interactions of plants with filamentous pathogens has been controversial for a long time.
Over the past decades, tremendous progress has been made in understanding the biological functions of apoplastic CDIPs in filamentous oomycetes and fungi, such as their contribution to pathogen virulence and being recognized by plant cells. These findings have greatly enriched our knowledge on the roles of apoplastic CDIPs as virulence factors and how plants detect them and mount a defense response. In this review, we summarize the latest advances in identifying apoplastic CDIP effectors in plant pathogenic oomycetes and fungi, and our current understanding of the dual roles of apoplastic CDIPs in plant-filamentous pathogen interactions.

APOPLASTIC CDIPS IDENTIFIED IN PLANT PATHOGENIC OOMYCETES AND FUNGI Apoplastic CDIPs Identified in Phytopathogenic Oomycetes
The purification and identification of extracellular proteins from phytopathogenic oomycetes that induce plant cell death began in the 1980s. Pernollet and colleagues purified three extracellular proteins, namely capsicein, cinnamomin and cryptogein, in culture filtrates of Phytophthora capsici, P. cinnamomi and P. cryptogea, respectively (Billard et al., 1988;Huet and Pernollet, 1989;Ricci et al., 1989). Determination of the amino acid sequences of these proteins led to definition of a novel protein family, called elicitins (Derevnina et al., 2016). Over the past decades, building on advances in molecular biology and genome sequencing, significant progress has been made in identifying extracellular proteins from phytopathogenic oomycetes that induce cell death in plants. To date, over 62 apoplastic CDIPs have been identified in 16 oomycete species (Table 1). While a few identified oomycete apoplastic CDIPs belong to PcF toxin, pectate lyase and glycoside hydrolase (GH) families or with no conserved domains, the majority are elicitins or Nep1 (necrosis-and ethylene-inducing peptide 1)-like proteins (NLPs) (Tables 1, 3).

Apoplastic CDIPs Identified in Phytopathogenic Fungi
Similarly, the availability of genome sequences of fungi has led to a rapid identification of apoplastic CIPDs from phytopathogenic fungi. To date, over 115 apoplastic CDIPs have been identified in 27 fungal species, including biotrophic, hemibiotrophic and necrotrophic pathogens ( Table 2). The identified fungi apoplastic CDIPs include CFEM (common in fungal extracellular membrane)-containing proteins, ceratoplatanin proteins (CPPs), GHs, NLPs, cutinases, and pectate lyases, and some proteins with other domains or with no conserved domains (Tables 2, 3). Among the identified oomycete and fungus apoplastic CDIPs, GHs, NLPs, Pectate lyases, and VmE02 homologues are widely distributed across oomycetes and fungi (Tables 1-3). On the other hand, many CDIPs are oomycete-specific or fungi-specific. For example, elicitins and elicitin-like proteins are unique to oomycetes, whereas CFEMcontaining proteins and CPPs are unique to fungi (Table 3).

MAJOR PROTEIN FAMILIES OF OOMYCETE AND FUNGUS APOPLASTIC CDIPS CFEM Containing CDIPs
The CFEM domain, which contains eight conserved cysteines, is unique to fungi (Kulkarni et al., 2003;Zhang Z. N. et al., 2015). CFEM was first identified in a M. oryzae MAC1-interacting protein, ACI1 (Kulkarni et al., 2003). CFEM containing proteins are widely distributed in fungi (Zhang Z. N. et al., 2015). While some of CFEM containing proteins have been identified to have prominent roles in pathogenicity or virulence, such as MoPth11 and its ortholog FGRRES_16221 in Magnaporthe oryzae and Fusarium graminearum, respectively (Kou et al., 2017;Dilks et al., 2019), and CgCcw14, CgMam3 in Candida glabrata (Srivastava et al., 2014), several CFEM containing proteins have been identified to possess cell death-inducing activity.
The CFEM-containing protein BcCFEM1 from Botrytis cinerea, contains a CFEM domain at the N-terminus, and a glycosylphosphatidylinositol (GPI) anchored site at the C-terminus. BcCFEM1 was induced and expressed at high levels during early stages of infection on bean leaves. Targeted disruption of the BcCFEM1 gene reduced virulence, conidiation and stress tolerance in B. cinerea. Transient expression of BcCFEM1 in Nicotiana benthamiana leaves triggered obvious chlorosis (Zhu et al., 2017b), suggesting the involvement of BcCFEM1 in eliciting plant responses.
The M. oryzae genome harbors 19 proteins with the CFEM domain (Kou et al., 2017). Two CFEM-containing proteins  Ricci et al., 1989;Galiana et al., 1997;Mikes et al., 1997;Leborgne-Castel et al., 2008;Coursol et al., 2015;Kulik et al., 2015;Ptáčková et al., 2015;Starý et Huet et al., 1994;Kamoun et al., 1997Kamoun et al., , 1998Kanzaki et al., 2003;Huitema et al., 2005;Du et al., 2015 (Continued) Frontiers in Genetics | www.frontiersin.org MoCDIP2 and MoCDIP11 have been identified to induce cell in the non-host N. benthamiana and host rice cells Guo et al., 2019). MoCDIP2 contains a CFEM domain at the N-terminus, and a GPI-anchored site at the C-terminus, whereas MoCDIP11 contains only a CFEM domain at the N-terminus. Transient expression assays in rice protoplasts or N. benthamiana leaves revealed that the signal peptides that led the secretion of proteins, were required for cell death inducing activity of MoCDIP2 and MoCDIP11, indicating that both two effectors function in the apoplast Guo et al., 2019).
Over 22 elicitin and elicitin-like CDIPs that induce cell death in certain plant species such as Nicotiana species and some Brassicaceae cultivars have been identified from different Phytophthora pathogens (Table 1). Among the elicitin and elicitin-like CDIPs, β-cryptogein and INF1 have been widely studied for their roles in the interactions of Phytophthora pathogens with plants. Cryptogein induced necrosis on tobacco leaves, triggered SAR and enhanced disease resistance in tobacco. Upon the SAR induction, the expression of the plant extracellular S-like RNase NE gene and its RNase activity were highly up-regulated, indicating that NE possibly associated with the cryptogein-induced SAR (Galiana et al., 1997). Cryptogein promoted the movement of plant respiratory burst oxidase homologues (RBOHs) from the Golgi cisternae to the plasma membrane, which may play a fundamental role for ROS production (Noirot et al., 2014). Cryptogein induced production of ROS, which was differentially regulated by the sphingolipid long-chain bases (LCBs) and their phosphorylated derivatives (LCB-Ps) in tobacco cells (Coursol et al., 2015). In addition, cryptogein induced production of NO, partly dependent on the ROS-dependent pathway, indicating that the defense responses induced by cryptogein involving interaction of the NO and ROS signaling pathways (Kulik et al., 2015). INF1 induced cell death on tobacco and potato leaves with the necrotic activity higher than that of other a-elicitins, such as cacto, parasiticein, capsicein and cryptogein, but less than that of βcryptogein (Huet et al., 1994). INF1 induced the expression of chitinase, β-1,3-glucanase, phenylalanine ammonia-lyase (PAL), and PR1 genes, and rapid accumulation of H 2 O 2 in tobacco (Sasabe et al., 2000;Huitema et al., 2005). INF1 induced the expression of NbrbohB in N. benthamiana, and silencing of NbrbohA or NbrbohB led to a reduction and delay of the necrotic reaction triggered by INF1 (Yoshioka et al., 2003), suggesting that INF1-induced cell death was dependent on the ROS burst.
Elicitins are able to bind sterols, phospholipids or fatty acids, and transport them between biological membranes (Mikes et al., 1997). However, the lipid-binding ability did not influence elicitin-induced response in tobacco. Investigations based on the mutant proteins of cryptogein with limited abilities to bind sterols revealed that induction of ROS synthesis, cytosol acidification and cell death in tobacco cells were not correlated with the sterolbinding abilities of the cryptogein proteins (Dokládal et al., 2012;Ptáčková et al., 2015).
Elicitins do not induce cell death in tomato plants. However, elicitins could induce immune responses in tomato, and enhance plant resistance against Phytophthora spp., bacterial wilt disease, and powdery mildew (Picard et al., 2000;Kawamura et al., 2009;Starý et al., 2019). INF1 induced the expression of jasmonic acid (JA)-responsive PR-6, LeATL6 and LOX-E, and ET-responsive PR-2b and ERF2, but not SA-responsive PR-1a and PR-2a in tomato leaves (Kawamura et al., 2009). Consistently, Starý et al. (2019) showed that cryptogein induced defense responses without cell death in tomato through JA-and ET-signaling pathways, but not SA-signaling pathway. These results indicated that elicitins triggered different signaling pathways between tobacco and tomato.

NLP Family CDIPs
NLP family proteins are characterized by the presence of a common NPP1 (necrosis-inducing Phytophthora protein) domain. This family is widely distributed across taxa including oomycetes, fungi, and bacteria (Gijzen and Nürnberger, 2006). The first NLP protein, namely Nep1, was identified from culture filtrates of F. oxysporum (Bailey, 1995), and has been shown to induce necrosis in plants, such as Erythroxylum coca, Theobroma cacao and Arabidopsis, with activating PR gene expression, ROS production and other general defense response (Bailey et al., 2005;Bae et al., 2006). To date, over 39 NLP family CDIPs have been identified in phytopathogenic oomycetes and fungi. Among them, 20 NLP family CDIPs were from phytopathogenic oomycetes ( Table 1), and the remaining 19 NLP CDIPs were from phytopathogenic fungi ( Table 2).
NLP family CDIPs have been confirmed to induce cell death in dicot but not monocot plants. Among the identified NLP family CDIPs, PpNLP/NLP Pp , PsojNIP and PaNie 213 /NLP Pya have been extensively studied in the context of inducing cell death and immune responses in dicot plants. PpNLP/NLP Pp induced necrosis in Arabidopsis, and activated PR genes expression, ROS production, callose apposition (Fellbrich et al., 2002), as well as the posttranslational expression of mitogen-activated protein kinase and production of nitric oxide and phytoalexin camalexin, suggesting dual roles of PpNLP/NLP Pp as both toxin-like virulence factors and plant innate immunity triggers (Qutob et al., 2006). Determining and modeling of the structures of PaNie 213 /NLP Pya , PpNLP/NLP Pp , and a PccNLP protein from the phytopathogenic bacterium Pectobacterium carotovorum, revealed that NLPs displayed identical toxin folds which contributed to host infection and plant defense gene activation, suggesting that a common fold of the cytolytic toxin is required for both pathogen virulence and plant immunity activation (Ottmann et al., 2009). Böhm et al. (2014) further identified and characterized a pattern of 20 amino acid residues (nlp20) of cytotoxic NLPs that triggered immunity associated plant defenses in certain dicot plants.
Based on the finding that NLPs display a striking similarity to cytolytic sphingomyelin-binding actinoporins (Ottmann et al., 2009), glycosylinositol phosphorylceramide (GIPC) sphingolipids in eudicot plants were further shown to be bound by NLPs. The inositol phosphorylceramide in GIPCs was covalently bound to glucuronic acid and variable terminal hexoses which were different between eudicots and monocots. Eudicot GPICs typically carried two terminal sugars (series A), while monocots GIPCs bore three terminal sugars (series B) (Cacas et al., 2013). The absence of series A GIPCs lead to insensitivity to NLPs of monocot plants. Consistently, Arabidopsis mutants with altered GIPC composition suffered less cell death than the wild type upon NLP infiltration (Lenarčič et al., 2017). These results thus explained host specificity of cell death induced by NLPs in eudicot plants (Van den Ackerveken, 2017).

Cell Wall-Degrading Enzyme Family CDIPs
Phytopathogenic oomycetes and fungi secrete a large amount of effector proteins related to plant cell wall degradation, such as enzymes to degrade cellulose, xylan, pectin, etc. (Kubicek et al., 2014). Certain cell wall-degrading enzymes (CWDEs) have been proved to be associated with pathogen virulence, and some induced cell death and trigged defense responses in plants. CWDE family CDIPs have been identified in carbohydrate esterase (CE5), glycoside hydrolase (GH) and polysaccharide lyase (PL) families (Tables 1-3).
With functions to degrade plant cuticle or suberin polymers, the CE family cutinases has been associated with important roles in filamentous pathogen-plant interactions. Two cutinase family CDIPs, SsCut1 and VdCUT11 have been identified in S. sclerotiorum and V. dahliae Gui et al., 2018), respectively. SsCut1 induced cell death in both dicot and monocot species and activated plant resistance against S. sclerotiorum, P. nicotianae and Phytophthora sojae . Further, SsCut1induced cell death along with stomatal closure, ROS burst and NO production, was suppressed by silencing of a C 2 H 2 -type zinc finger gene NbCZF1 in N. benthamiana, showing that SsCut1-triggered defense could be mediated by NbCZF1-ROS-NO pathway . VdCUT11 induced cell death and defense responses in N. benthamiana, cotton, and tomato plants, and the enzymatic activity was required for its cell death-inducing activity (Gui et al., 2018).
GHs hydrolyze the glycosidic bond between carbohydrates or between a carbohydrate and a noncarbohydrate moiety through acid catalysis (Zhao et al., 2013). About 18 GH domaincontaining CDIPs have been identified ( Table 3). The GH family CDIPs induced cell death as well as defense responses in host and nonhost plants. For example, BcXyn11A induced ROS burst, electrolyte leakage, cytoplasm shrinkage and PR gene expression, and these effects were dependent on its short 25-residue peptide (Frías et al., 2019). BcXYG1 triggered pattern-triggered immunity (PTI) response and systemic resistance in bean (Zhu et al., 2017a). PsXEG1 induced disease resistance in N. benthamiana and soybean (Ma et al., 2015). BcGs1 induced systemic resistance in tobacco and tomato against B. cinerea, Phytophthora syringae and tobacco mosaic virus . Furthermore, BcGs1 triggered ROS burst, PAL and peroxidase (POD) enzyme activity, and lignin accumulation in tomato (Yang et al., 2018a). Many of the GH family CDIPs have been shown to possess hydrolase activity. While hydrolase activity of BcPG2 was required for its cell death induction (Kars et al., 2005), the enzymatic activity of most identified GH family CDIPs was not necessary for cell death inducing activity. For example, the cell death inducing activity of Xyn11A, PsXEG1, BcXYG1, VdEG1 and VdEG3 was independent of their enzymatic activity (Brito et al., 2006;Ma et al., 2015;Gui et al., 2017;Zhu et al., 2017a).
The PL family pectate lyases play a critical role in pectin degradation. Four pectate lyases PcPL1, PcPL15, PcPL16 and PcPL20 in P. capsici, and one pectate lyase VdPEL1 in V. dahliae have been identified to have cell death inducing activity in plants (Fu et al., 2015;Yang et al., 2018d). PcPL1, PcPL15, PcPL16 and PcPL20 were highly expressed in P. capsici during infection of pepper, and transient expression of the four PcPLs induced severe cell death in pepper leaves (Fu et al., 2015). VdPEL1 induced cell death in N. benthamiana, tomato, soybean and cotton, and triggered defense responses and systemic resistance to B. cinerea and V. dahliae in N. benthamiana and cotton plants. Furthermore, the enzymatic activity was found to be necessary for cell death-inducing activity (Yang et al., 2018d).
Besides, a SGNH hydrolase subfamily protein BcXyl1 was identified to induce cell death in N. benthamiana, tomato, soybean and cotton (Yang et al., 2018c). BcXyl1 exhibited xylanase activity, but its cell death inducing activity was independent of the enzymatic activity. BcXyl1 triggered plant PTI responses with a pattern of 26-amino acid peptide.

CDIP Families With Other Conserved Domains or Without Conserved Domain
Besides the above-mentioned CDIP families, a number of apoplastic CDIPs contain other conserved domains or no conserved domain (Tables 1, 2), indicating the incredible diversity of apoplastic CDIPs secreted by oomycetes and fungi. On the other hand, many of these apoplastic CDIPs are widely distributed across microbial taxa or different pathogen species and the homologs can induce cell death and defense responses in different plant species, indicating that recognition of these proteins is evolutionarily conserved. For example, the Valsa mali small cysteine-rich protein VmE02 induces cell death in N. benthamiana, tomato, pepper, Arabidopsis and apple and enhances resistance in N. benthamiana against S. sclerotiorum and P. capsic . VmE02 is widely conserved across oomycete and fungal species, and the homologs can induce cell death in N. benthamiana. Similarly, the Colletotrichum orbiculare NIS1 and its homologs from Colletotrichum higginsianum and Fusarium virguliforme (Yoshino et al., 2012;Chang et al., 2016), and the Rhynchosporium commune RcCDI1 and its homologues from M. oryzae, Neurospora crassa and Zymoseptoria tritici (Franco-Orozco et al., 2017), induced cell death in N. benthamiana. These results clearly supported that, although the physiological properties remain unknown, these apoplastic CDIPs are recognized as conserved patterns that induce defense responses in plants.

APOPLASTIC CDIPS CONTRIBUTE TO PATHOGEN VIRULENCE
Phytopathogenic oomycetes and fungi initially colonize in the plant apoplast or extracellular space, and subsequently penetrate host cells. Therefore, the apoplastic effectors secreted by oomycetes and fungi are likely the primary weapons of filamentous plant pathogens. Despite the diversity of lifestyles as biotrophic, hemibiotrophic or necrotrophic, filamentous oomycetes and fungi secrete a high number of apoplastic CDIPs. Biotrophic pathogens feed on living plant cells (Giraldo and Valent, 2013). Whether apoplastic CDIPs of biotrophic pathogens actually cause plant cell death or function as important virulence factors during a natural infection remain to be determined (Tables 1, 2). In contrast, necrotrophic pathogens thrive on dead host tissues and take advantage of CDIP-triggered plant cell death. For example, NLP-triggered necrosis could aid infection by necrotrophic pathogens (Van den Ackerveken, 2017). Hemibiotrophic pathogens combine a biotrophic phase in early stages with a necrotrophic phage during later infection stages. Many apoplastic CDIPs of hemibiotrophic pathogens have been found to be highly expressed at late infection stages, suggesting that they contribute to the necrotrophic growth of hemibiotrophic pathogens (Qutob et al., 2002;Kelley et al., 2010).
Functional analysis based on overexpression, deletion or silencing of genes encoding apoplastic CDIPs have functionally proved many of them as important virulence factors in hemibiotrophic and necrotrophic pathogens (Tables 1, 2). Among the 62 identified oomycete apoplastic CDIPs, 12 have been proven to function as virulence factors that are required for pathogenicity or contribute to virulence. The P. cactorum SCR96 belongs to the PcF toxin family. Silencing of the scr96 gene in Phytophthora cactorum caused loss of pathogenicity on host plants, indicating that SCR96 is required for pathogenicity (Chen et al., 2016). In P. capsici, silencing of the genes encoding NLP-like proteins PcNLP2, PcNLP6 or PcNLP14, and the genes encoding pectate lyases PcPL1, PcPL15, PcPL16 or PcPL20 caused significantly reduced virulence on pepper (Feng et al., 2014;Fu et al., 2015), demonstrating the important roles of these apoplastic CDIPs in pathogen virulence. In Phytophthora palmivora, three fractions isolated from culture filtrates including high-molecular-weight glycoprotein, broadmolecular-weight glycoprotein and 42-kDa glycoprotein were observed to promote P. palmivora infection of rubber tree leaves (Pettongkhao and Churngchow, 2019). However, the virulence role of these three glycoproteins remains to be genetically confirmed. The P. sojae XEG1, a GH12 family CDIP, functions as a major virulence factor during infection. Both silencing and overexpression of the PsXEG1 gene in P. sojae severely impaired virulence (Ma et al., 2015). Interestingly, P. sojae also secretes a PsXLP1 (PsXEG1-like) apoplastic effector with a truncated GH12 domain functioning as a decoy to shield XEG1-mediated virulence (Ma et al., 2017).
Among the 115 identified fungi apoplastic CDIPs, 16 have been proven to be required for pathogenicity or contribute to virulence of both hemibiotrophic and necrotrophic pathogens. In B. cinerea, targeted deletion of the genes encoding BcCFEM1 (Zhu et al., 2017b), BcSpl1 (Frías et al., 2011), BcXyl1 (Yang et al., 2018c), or BcXyn11A (Brito et al., 2006) caused severely compromised virulence, suggesting that these CDIPs were important virulence factors of B. cinerea. Overexpression and deletion of BcXYG1 did not significantly affect B. cinerea infection on bean leaves. However, the BcXYG1 overexpression strains produced significantly earlier and more intense local necrosis, suggesting that BcXYG1 contributes to the establishment of infection in early stages (Zhu et al., 2017a). The C. gloeosporioides CgCP1 belongs to the CPP family. Knock-out of CgCP1 in C. gloeosporioides significantly reduced infection on rubber tree leaves . Similar to BcSpl1 and CgCP1, the CPP family CIDPs FocCP1, SsCP1 and VdCP1 function as important virulence factors. Deletion of FocCP1 in F. oxysporum (Liu et al., 2019), SsCP1 in S. sclerotiorum (Yang et al., 2018b), or VdCP1 in V. dahliae (Zhang et al., 2017a) caused significantly reduced virulence of pathogens on hosts banana, Arabidopsis or cotton, respectively. In M. oryzae, deletion of mohrip1, or mohrip2 remarkably compromised fungal virulence on rice Nie H. Z. et al., 2019). MoHrip1 belongs to the Alt a 1 (AA1) family . Similarly, the V. dahliae PevD1, an AA1 family CDIP, is required for full virulence of V. dahliae on hosts . In addition to VdCP1 and PevD1, VdCUT11, VdEG1, VdEG3, VdNLP1, VdNLP2, and VdPEL1 have been shown to play important roles in virulence of V. dahliae. Targeted deletion of VdCUT11 (Gui et al., 2018), VdEG1, VdEG3 (Gui et al., 2017), or VdPEL1 (Yang et al., 2018d) significantly compromised virulence of V. dahliae on cotton plants. Interestingly, while both VdNLP1 and VdNLP2 appear to be dispensable for V. dahliae infection in cotton plants , VdNLP1 as well as VdNLP2 deletion strains were found to be significantly less pathogenic on tomato and Arabidopsis (Santhanam et al., 2013), demonstrating the functional diversification of the two NLP family CDIPs in virulence of V. dahliae.
Pathogenicity assays also revealed that many apoplastic CDIPs were not required for fungal pathogenicity or virulence. Targeted deletion of the genes encoding these proteins did not impair the virulence of pathogens ( Table 2). One possibility why these apoplastic CDIPs were dispensable for virulence could be due to the functional redundancy with other apoplastic effectors (Yoshino et al., 2012;Guo et al., 2019).

RECOGNITION OF APOPLASTIC CDIPS IN PLANTS: CELL SURFACE RECEPTORS AND DOWNSTREAM COMPONENTS
In recent years, there have been breakthroughs in the identification of cell surface receptors recognizing apoplastic CDIPs. By screening T-DNA insertion mutants and natural accessions of Arabidopsis, Albert et al. (2015) identified T-DNA insertion alleles of RLP23 and Arabidopsis accessions carrying a frameshift mutation of RLP23 that were insensitive to the conserved nlp20 pattern found in most NLPs. RLP23 encodes a leucine-rich repeat-receptor-like protein (LRR-RLP), which binds extracellularly to nlp20, thereby mediating NLP-elicited immune response in Arabidopsis (Albert et al., 2015). Transgenic potato plants expressing RLP23 displayed enhanced resistance to oomycete and fungal pathogens, such as P. infestans and S. sclerotiorum (Albert et al., 2015), further supporting that RLP23 confers protection to oomycete and fungal pathogens. More recently, RXEG1 (Response to XEG1), an LRR-RLP that specifically recognizes the GH12 family CDIP XEG1, has been identified from N. benthamiana through a high-throughput virus-induced gene silencing (VIGS) screen . RXEG1 interacts with XEG1 by the extracellular LRR domain in the apoplast, and regulates XEG1-induced plant cell death and immune responses.
Using a genetic mapping approach, an ELR (elicitin response) gene has been identified from a population derived from the cross of Solanum microdontum genotype mcd360-1 (responds to INF1 with a cell death response) with S. microdontum ssp. gigantophyllum gig714-1 (does not respond to INF1) (Du et al., 2015). ELR encodes an LRR-RLP, and ELR mediates extracellular recognition of a broad range of elicitins exhibiting relatively low sequence similarity, suggesting that ELR recognizes elicitins most likely based on domain similarity but not a small conserved peptide. Moreover, cultivated potato transformed with the ELR gene exhibited enhanced resistance to Phytophthora infestans. Overall, the results suggested ELR as a potential cell surface receptor to mediate response to elicitins. However, the physical association of ELR with INF proteins remains unclear (Du et al., 2015). Interestingly, two intracellular proteins, a lectin-like receptor kinase (NbLRK) and a pepper calcium-binding protein (SRC2-1), have been shown to interact with P. infestans INF1 (Kanzaki et al., 2008) and P. capsici PcINF1 , and mediate P. infestans INF1 or PcINF1-induced cell death, respectively. These results correspond with the speculation that elicitins could be possibly transported into plant cells through clathrin-mediated endocytosis (Leborgne-Castel et al., 2008). Hence, it would appear that plants possess multiple mechanisms to recognize elicitins.

CONCLUDING REMARKS AND PERSPECTIVES
An emerging number of studies have shown that many apoplastic CDIPs are required for pathogenicity, or contribute to the virulence of hemibiotrophic and necrotrophic pathogens, demonstrating the important role of these apoplastic CDIPs as essential virulence factors. On the contrary, apoplastic CDIPs were shown to elicit plant defense responses. Over the past decades, studies have documented that many apoplastic CDIPs are recognized by plants as PAMPs, being able to trigger PR gene expression, phytoalexin synthesis, MAPK phosphorylation, SA-, JA/ET-signaling pathways, as well as resistance against pathogens. The recent findings of extracellular recognition of NLPs, elicitins and XEG1 by plant RLPs, RLP23, ELR and RXEG1 (Albert et al., 2015;Du et al., 2015;Wang Y. et al., 2018), respectively, have greatly advanced our understanding of the roles of apoplastic CDIPs in plant-pathogen interactions. More importantly, the identification of receptors for apoplastic CDIPs provides valuable gene resources for engineering crops with broad and durable disease resistance (Albert et al., 2015;Du et al., 2015).
Currently, the majority of apoplastic CDIPs have been characterized based on demonstrations in dicot plants, especially based on transient expression in N. benthamiana, even many apoplastic CDIPs were identified from monocot pathogens. Particularly, recognition receptors and downstream components that have been identified remain restricted to dicot plants. Given the paramount importance of monocot cereal plants, such as rice, wheat and maize, as staple crops, it would be important to determine recognition of apoplastic CDIPs in monocot hosts, which could help engineer monocot cereal crops with broadspectrum resistance against filamentous pathogens.