Deciphering host manipulation strategies: Exploring Effector-target interactions in necrotrophic pathosystem

Introduction

Plant-fungal interactions are categorized into different types such as biotrophic, hemibiotrophic, and necrotrophic interactions, based on the pathogen's feeding behavior. Much of the scientific exploration in genetics and molecular biology has concentrated on the classical biotrophic feeding strategy. In this strategy, pathogens produce effector molecules that aid in colonization by manipulating the host. In response, plants have developed surveillance mechanisms to detect pathogen-related molecular patterns (Dodds and Rathjen, 2010) [11] , (Thomma et al., 2011) [82] , (Toruno et al., 2016) [85] . These mechanisms include pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs), and effector-triggered immunity (ETI), which involves the recognition of pathogen-produced effector molecules. ETI often engages host resistance genes, particularly those encoding proteins with nucleotide-binding and leucine-rich repeat domains (NLR proteins). These conceptual models have significantly contributed to our understanding of host-pathogen interactions, exemplified by systems like the Melampsora lini-flax pathosystem proposed by H.H. Flor, which introduced the gene-for-gene hypothesis (Flor, 1942) [23] , (Flor, 1955) [22] , (Flor, 1971) [21] . To thrive and reproduce, pathogens must navigate around, inhibit, or influence the plant's innate defense mechanisms effectively. These mechanisms typically involve activating genes responsible for defense responses, increasing the production of pathogenesis-related (PR) proteins like chitinase and antimicrobial compounds, generating harmful levels of reactive oxygen species (ROS) through oxidative bursts, initiating defense pathways such as the phenylpropanoid pathway, and triggering the hypersensitive response (HR), a process that leads to controlled cell death in localized areas. While all pathogens must manipulate elements of the defense response, necrotrophic pathogens have not been easily categorized within the established models of plant immunity designed for biotrophs. This article examines three necrotrophic pathogens -Pyrenophora tritici-repentis, Parastagonospora nodorum, and Zymoseptoria tritici -constituting the foliar disease complex in wheat. It explores how these pathogens, despite inducing similar necrotic symptoms, have evolved both shared and distinct strategies for colonization aimed at achieving the ultimate goal of reproduction (sporulation). The discussion centers on the effectors produced by these pathogens and the corresponding receptors/targets in the host that mediate these necrotrophic interactions, leading to the development of three distinct wheat diseases of global significance. The review underscores the notion that each pathogen has independently evolved in concert with its specific host, resulting in a complex sequence of precisely timed interactions driven by the ongoing evolution of hostpathogen relationships.

Pyrenophora tritici-repentis -Wheat Pathosystem Infection Process

Tan spot, a significant disease in wheat production worldwide, is caused by P. tritici-repentis (Faris et al., 2013) [18] . Early studies utilized light microscopy to observe pathogen germination, penetration, and colonization patterns (Larez et al., 1986) [45] , (Loughman and Deverall, 1986) [53] . These investigations revealed the formation of appressorium-like structures on epidermal cells, followed by penetration pegs entering these cells and developing vesicles. Secondary hyphae emerged from these vesicles and infiltrated the mesophyll layer within 24 hours, colonizing intercellularly without penetrating mesophyll cells. Interestingly, penetrated epidermal cells exhibited dark staining indicative of cell death once secondary hyphae reached the mesophyll layer (Loughman and Deverall, 1986) [53] . By 72 hours, the fungus disrupted mesophyll cell membranes beyond hyphal growth (Larez et al., 1986) [45] , suggesting the secretion of molecules inducing programmed cell death ahead of mycelial advancement. Macroscopically, it induces lens-shaped lesions with necrotic centers and chlorotic borders. Initially, these symptoms were categorized as tan necrosis and extensive chlorosis (Lamari and Bernier, 1989a, 1989b) [40,41] . Later, a genetically distinct symptom, localized chlorosis, was identified. The diverse symptomatology led to the classification of eight races based on combinations of necrotic and chlorotic symptoms (Lamari et al., 1995, 2003) [42,43] . These symptoms were linked to necrotrophic effectors Ptr ToxA, Ptr ToxB, and Ptr ToxC (Ciuffeti et al., 2010), (Lamari and Strelkov, 2010) [44] . Susceptibility to each virulence component was genetically dominant, indicating an inverse gene-for-gene interaction (Lamari and Strelkov, 2010) [44] .

Effector -Susceptibility factor Interaction

Tomas and Bockus (1987) [83] demonstrated that P. triticirepentis culture filtrates induced necrotic symptoms in susceptible wheat lines but not in resistant ones. The necrosis-inducing component was identified as a small secreted protein (Balance et al., 1989), (Tomas et al., 1990) [84] , later confirmed to be encoded by the single-copy ToxA gene, the first proteinaceous host-selective toxin found in any plant pathogen. Investigations into host metabolism and signaling revealed their necessity for ToxA-induced cell death, showcasing the pathogen's use of ToxA to trigger host-controlled programmed cell death, a mechanism typically associated with resistance to biotrophic pathogens (Kwon et al., 1998) [39] . An Arg-Gly-Asp (RGD) motif in Ptr ToxA was found to interact with an extracellular receptor, essential for inducing necrosis, as confirmed by mutagenesis studies. Ptr ToxA was internalized into sensitive wheat mesophyll cells, targeting chloroplasts and disrupting photo system processes, leading to lightdependent reactive oxygen species (ROS) accumulation and cell death (Manning et al., 2009) [54] . The susceptibility gene Tsn1 was identified as a dominant gene conferring sensitivity to ToxA, indicating its role in susceptibility to ToxA-producing P. tritici-repentis isolates (Faris et al., 1996) [14] . Transcriptional profiling studies revealed upregulation of defense-associated genes involved in signal transduction, phenylpropanoid pathway regulation, cell wall lignifications, ROS signaling, and pathogenesis-related (PR) proteins in ToxA-treated plants, reflecting the ToxA-Tsn1 interaction and resulting in sensitivity/susceptibility responses. Ptr ToxB, a newly identified necrotrophic effector, was partially purified and characterized (Strelkov et al., 1998) [77] . It was found to induce chlorosis in a light-dependent manner by degrading chlorophyll through photo oxidation. This effector, consisting of a 6.61-kDa heat-stable protein, is encoded by a multicopy gene with a 261-bp open reading frame producing a 64-amino acid mature protein (Strelkov et al., 1999) [76] , (Martinez et al., 2001) [57] . Its increased copy number and expression are speculated to correlate with enhanced virulence (Martinez et al., 2004) [56] , (Strelkov and Lamari, 2003) [79] , (Strelkov et al., 2002) [78] . Due to its stability and cysteine content, Ptr ToxB is believed to localize in the apoplast, showing resistance to protease digestion and high tolerance for apoplastic conditions. Fluorescent labeling demonstrated its apoplastic localization without internalization into cells (Figueroa et al., 2015) [20] . Structural analysis revealed similarities between Ptr ToxB and AvrPiz-t, an effector from Magnaporthe oryzae (Nyarko et al., 2014) [62] . This structural resemblance suggests Ptr ToxB might target biotrophic resistance mechanisms, inducing programmed cell death (PCD) and susceptibility, akin to AvrPiz-t in rice. The Ptr ToxB-Tsc2 interaction, which accounts for a significant portion of phenotypic variation, operates in an inverse gene-for-gene manner, with Tsc2 mapped to wheat chromosome 2B (Friesen and Faris, 2004). Transcriptional analysis of host responses to Ptr ToxB infiltration, compared to Ptr ToxA, revealed similar defense gene activation but with a delayed response (Pandelova et al., 2012) [66] . Less research has focused on the Ptr ToxC-Tsc1 interaction. Genetic assessment showed susceptibility to a chlorosisinducing P. tritici-repentis strain, with a major QTL located on wheat chromosome 1A (Faris et al., 1997) [15] . Ptr ToxC, unlike Ptr ToxA and Ptr ToxB, is a small, polar molecule (Effertz et al., 2002) [14] . Sensitivity to Ptr ToxC also mapped to chromosome 1A, the same locus associated with susceptibility to Ptr ToxC-producing races (Effertz et al., 2001) [13] , (Faris et al., 1997) [15] . Overall, the interactions between P. tritici-repentis and wheat necrotrophic effectors highlight diverse mechanisms of programmed cell death (PCD) in the pathogen's life cycle. Ptr ToxA, likely horizontally transferred from P. nodorum, rapidly induces necrosis by targeting an NLR resistance-like protein (Friesen et al., 2006) [30] . Ptr ToxB, structurally similar to avirulence effectors from M. oryzae, induces slower chlorosis. Ptr ToxC, a low-molecular-weight molecule, triggers extensive chlorosis. Despite their differences, all three effectors serve as virulence factors for the pathogen's necrotrophic life cycle.

Parastagonospora nodorum -Wheat Pathosystem Infection Process

Septoria nodorum blotch (SNB), caused by P. nodorum, and has posed a significant threat to foliar health globally (Faris and Friesen, 2020) [16] , (Mehra et al., 2019) [59] , (Murray and Brennan, 2009) [61] , (Oliver et al., 2012) [64] , (Singh et al., 2016) [74] , (Xu et al., 2004) [87] . This wheat-specialized necrotrophic pathogen induces cell death in susceptible wheat lines to complete its life cycle. Studies have documented both direct epidermal and stomata penetration by P. nodorum (Baker and Smith, 1978) [2] , (Bird and Ride, 1981) [5] , (O'Reilly and Downes, 1986) [63] . The first indication of light-dependent repression of necrotrophic effector activity was observed by (Baker and Smith, 1978) [2] , highlighting the significance of environmental cues in disease progression. Upon penetration, epidermal cells undergo rapid collapse and lignifications as mycelia advance into the mesophyll layer (Baker and Smith, 1978) [2] , (Bird and Ride, 1981) [5] . Mesophyll cells undergo shape alterations, lignifications, and cytoplasmic degeneration, ultimately leading to complete collapse. Chloroplast deterioration and reduced photosynthetic rates indicate early host manipulation by the pathogen. Infection progresses rapidly, with mycelia reaching the mesophyll layer within days, followed by severe cellular collapse and eventual colonization. P. nodorum secretes a variety of cell walldegrading enzymes (CWDEs) believed to aid in host tissue penetration (Lehtinen, 1993) [47] , (Margo, 1984). While these enzymes display general toxicity to cell wall components, they lack host selectivity. Crude extracts containing toxic compounds from P. nodorum demonstrated selective action on wheat embryos, indicating the presence of necrotrophic effectors. This method was employed to differentiate resistance levels in breeding material, providing early evidence of P. nodorum's secreted necrotrophic effectors (Wicki et al., 1999) [86] .

Effector -Susceptibility factor Interaction

SnTox1, a necrotrophic effector produced by P. nodorum, targets the wheat susceptibility gene Snn1 to facilitate its life cycle. This small secreted protein induces necrosis within 72 hours in wheat genotypes carrying Snn1, with sensitivity mapped to chromosome 1BS. The SnTox1-Snn1 interaction, accounting for up to 58% of disease variation, triggers host defense responses, including an oxidative burst, PR protein upregulation, and programmed cell death (PCD), promoting susceptibility (Liu et al., 2004a, 2004b) [50, 52] . SnTox1 expression peaks at 3 days, coinciding with sporulation initiation, and its infiltration or inoculationinduced necrosis is light-dependent (Liu et al., 2016, 2012) [48, 49] . Unlike other effectors, SnTox1 induces necrosis both upon infiltration and spraying onto the leaf surface, suggesting involvement in early penetration. SnTox1's prevalence across isolates suggests additional roles beyond Snn1 targeting (McDonold et al., 2013), (Richards et al., 2019) [68] . Structural similarity to plant chitin-binding proteins allows SnTox1 to bind chitin, protecting P. nodorum from wheat chitinase upregulated during SnTox1-Snn1 interaction (Liu et al., 2012) [49] . Snn1 encodes a WAK, a pattern recognition receptor (PRR) recognizing damage-associated molecular patterns (DAMPs), implying SnTox1's hijacking of a PRR-mediated defense pathway (Shi et al., 2016) [71] . TaMAPK3 upregulation upon SnTox1 inoculation suggests activation of plant immune responses, further validated by yeast two-hybrid experiments demonstrating SnTox1-Snn1 interaction. This interaction represents the first instance of a necrotrophic pathogen targeting a PRR, hijacking host defenses to facilitate its life cycle. ToxA, the first proteinaceous necrotrophic effector identified, holds significance for three wheat pathogens (Ciuffetti et al., 2010) [9] , (Friesen et al., 2018) [28] , (McDonald et al., 2018) [58] . Initially discovered in P. triticirepentis, its mode of action has been extensively studied. A homolog, SnToxA, was found in the P. nodorum genome, sharing high homology with PtrToxA. Diversity studies suggest ToxA originated in P. nodorum and transferred horizontally to P. tritici-repentis (Friesen et al., 2006) [30] . The ToxA-Tsn1 interaction confers susceptibility to Septoria nodorum blotch in both seedlings and adult plants (Faris and Friesen, 2009, 2020) [17,16] , (Friesen et al., 2009, 2006) [24,30] . While Tsn1 contains domains typical of classical resistance genes, its direct interaction with ToxA or related proteins is unclear (Tai et al., 2007) [81] . However, Tsn1 appears involved in ToxA internalization. This discovery underscores how necrotrophic pathogens exploit host defense mechanisms for their benefit, highlighting a shift from classical gene-for-gene resistance to susceptibility in multiple pathosystems. SnTox3, a proteinaceous necrotrophic effector, targets the wheat gene Snn3 (Friesen et al., 2008) [26] , initially identified on chromosome 5B and later found on 5D (Zhang et al., 2011) [88] . This interaction significantly impacts disease susceptibility, ranging from 17% to 52% at the seedling stage and up to 24% in adult plants (Friesen et al., 2008) [26] . Allelic variation in Snn3-B1 may influence sensitivity to SnTox3, suggesting diverse disease outcomes (Shi et al., 2016) [73] . Cloning Snn3-D1 revealed S/TPK and MSP domains (Zhang et al., 2021) [89] , both essential for SnTox3 sensitivity, highlight the broad arsenal of hosttargeted genes by P. nodorum for inducing programmed cell death (PCD). The SnTox3 gene encodes a 693-bp intronfree sequence with no known homologs, expressed early during infection (Liu et al., 2009) [51] . Its pro-sequence, cleaved by Kex2, forms critical disulfide bonds, typical of apoplastic effectors. SnTox3 interacts with TaPR-1 proteins (Breen et al., 2016) [7] , including the CAPE1 defense signaling peptide, enhancing susceptibility to SnTox3producing isolates. SnTox3 disrupts host defense by preventing TaCAPE1 release from TaPR1, suppressing host immunity. Host responses to SnTox3 infiltration involve differential regulation of defense pathways, PCD-related processes, and phenylpropanoid accumulation, illustrating P. nodorum's strategy of hijacking defense mechanisms to complete its pathogenic life cycle (Sung et al., 2021) [80] . The interactions SnTox2-Snn2, SnTox6-Snn6, and SnTox7-Snn7 were identified and studied independently through host target mapping and sensitivity to necrotrophic effector activity (Friesen et al., 2007) [29] , (Gao et al., 2015) [32] , (Shi et al., 2015) [72] . These interactions contributed quantitatively to disease variation, with Snn2, Snn6, and Snn7 mapping to different loci. The proteins SnTox2, SnTox6, and SnTox7 were all small secreted proteins. A genomic study of P. nodorum revealed a single virulence locus associated with Snn2 and Snn6, indicating that SnTox2 and SnTox6 target both genes. Further analysis confirmed that Snn2, Snn6, and Snn7 are all targeted by the same protein, SnTox267. Snn2 and Snn6 were found to be complementary genes in the same pathway required for sensitivity to SnTox267. Light dependency varied between the Snn2/Snn6 and Snn7 pathways, suggesting that SnTox267 targets separate pathways to facilitate the pathogen's life cycle (Richards et al., 2022) [67] . A tetraploid wheat mapping population was utilized to uncover the SnTox5-Snn5 interaction (Friesen et al., 2012) [25] , where SnTox5, a small heat-stable protein, targeted Snn5, inducing necrosis to benefit the pathogen. This interaction was light-dependent and contributed significantly to disease variation, particularly when combined with other necrotrophic effectors like SnToxA-Tsn1, showing synergistic effects. SnTox5 encoded a 217-aa small secreted protein with six cysteine residues crucial for its activity, with a predicted signal peptide and pro-sequence aiding in folding (Kariyawasam et al., 2021) [34] . Besides targeting Snn5, SnTox5 aided mesophyll colonization, even without the presence of the Snn5 gene. Overall, P. nodorum employs various small secreted proteinaceous necrotrophic effectors, such as SnToxA (Friesen et al., 2006) [30] , SnTox1 (Liu et al., 2012) [49] , SnTox3 (Liu et al., 2009) [51] , SnTox4 (Abeysekara et al., 2009) [1] , SnTox267 (Richards et al., 2022) [67] , and SnTox5 (Kariyawasam et al., 2021) [34] , to target dominant susceptibility genes, inducing classical resistance responses that favor the pathogen's advantage. These host genes include an NLR (Faris et al., 2010) [19] , a WAK (Shi et al., 2016) [71] , and an S/TPK-MSP (Zhang et al., 2021) [89] , each triggering typical resistance responses.

Zymoseptoria tritici -Wheat Pathosystem

Infection process Z. tritici, the causative agent of septoria tritici blotch (STB), has been labeled a hemibiotroph, though it lacks biotrophic structures during the asymptomatic phase. Instead, it's proposed as a latent necrotroph, remaining undetected until triggering necrosis during symptomatic phases to access nutrients (Kema et al., 1996) [37] , (Keon et al., 2007) [38] , (Sanchez-Vallet et al., 2015) [70] . Infections begin with germination and stomata penetration as early as 12 hours post inoculation (hpi), progressing to mesophyll colonization by 48 hpi. From 2 to 8 days post inoculation (dpi), mycelia continue intercellular colonization while evading detection. Mesophyll cell collapse starts at 8 dpi, with rapid cell death increasing until 12 dpi, followed by heightened mycelial growth from host nutrient release. Incompatible interactions show attempted penetration and limited mesophyll colonization without HR (Kema et al., 1996) [37] .

Effector Proteins in Z. tritici: Manipulating Host Defenses

Effector proteins in plant-pathogen interactions manipulate host defenses, aiding pathogen survival and reproduction. In the case of Z. tritici, effector-like proteins are secreted during asymptomatic colonization to evade detection and host defenses (Haueisen et al., 2019) [33] , (Palma-Guerrero et al., 2017) [65] . Mg1LysM and Mg3LysM, two upregulated genes during asymptomatic infection, bind chitin and protect against host enzymes (Marshall et al., 2011) [55] . While disruption of Mg1LysM had no impact on virulence, ΔMg3LysM mutants failed to colonize leaves, preventing necrosis and sporulation. Additionally, Mg3LysM delays the wheat defense response, crucial for pathogen colonization. Wheat homologs of CERK1 and CEBiP, involved in PTI, were identified, with their silencing allowing ΔMg3LysM mutants to regain pathogenicity, highlighting Mg3LysM's role in evading wheat PTI (Lee et al., 2014) [46] .

Unraveling Complexity in Z. tritici-Wheat Interactions

Simpler interactions can lead to progress due to easy understanding. In the Z. tritici-wheat system, numerous resistance genes (Stb genes) and quantitative trait loci (QTL) have been identified, suggesting a complex relationship. Qualitative and quantitative aspects, frequently combined, affect the virulence of Z. tritici (Kema et al., 2000) [36] , (Meile et al., 2018) [60] , (Stewart et al., 2018) [75] . ZtNIP1, a protein secreted by Z. tritici, triggers necrosis in wheat plants, potentially aiding in the transition from asymptomatic to necrotic phases of the pathogenic cycle by inducing programmed cell death (PCD). This suggests that multiple necrosis-inducing proteins could be involved in triggering plant defense responses. Z. tritici populations were studied to pinpoint a single gene, AvrStb6, responsible for triggering avirulence in wheat plants carrying the Stb6 resistance gene (Brading et al., 2002) [6] , (Chartrain et al., 2005) [8] . AvrStb6, a small cysteine-rich protein, didn't directly interact with Stb6 but initiated an avirulence response (Kema et al., 2018) [35] , (Zhong et al., 2017) [90] . Stb6, identified as a wall-associated kinase (WAK) gene, didn't induce programmed cell death (PCD) upon interaction with AvrStb6 (Saintenac et al., 2018) [69] . Recent research revealed both major (qualitative) and minor (quantitative) loci influencing Z. tritici virulence across different wheat varieties. These effects were genotype-specific, with natural isolates showing various combinations of these loci. Avr3D1, a large-effect QTL, was identified as a host-specific avirulence factor targeted by the Stb7 resistance gene (Meile et al., 2018) [60] . Despite being present in all isolates, allelic variations allowed Z. tritici to evade recognition by Stb7, suggesting a fitness advantage. Avr3D1 expression peaks during the asymptomatic phase, aiding early colonization and evasion of host recognition. Table 1 presents characterized wheat genes/proteins in the pathosystem with Pyrenophora triticirepentis, Parastagonospora nodorum, and Zymoseptoria tritici, while Table 2 details characterized pathogen genes/proteins in the same wheat pathosystem. [3] , (Ciuffetti et al., 1997) [10] ToxB Induces chlorosis through targeting Tsc2 (Martinez et al., 2001) [57] , (Strelkov et al., 1999) [76]

Parastagonospora nodorum-wheat pathosystem

SnToxA

Necrotrophic effector directed at the NLR gene Tsn1 (Friesen et al., 2006) [30]

SnTox1

Necrotrophic effector directed at the WAK-like gene Snn1 (Liu et al., 2012) [49]

SnTox3

Necrotrophic effector directed both Snn3-B1 and Snn3-D1 (Liu et al., 2004) [50]

SnTox267

Necrotrophic effector directed at Snn2, Snn6, and Snn7 (Richards et al., 2022) [67]

SnTox5

Necrotrophic effector specifically targeting Snn5 (Kariyawasam et al., 2021) [34]

Zymoseptoria tritici-wheat pathosystem

ZtNIP1

Causes necrosis in Arabidopsis, yet its involvement in the Z. tritici-wheat pathosystem haven't been proven. (Ben M'Barek et al., 2015) [4] Avr3D1 Possibly involved in initial colonization but countered by Stb7, leading to resistance. (Meile et al., 2018) [60]

AvrStb6

Targeted by Stb6 to induce early defense (Kema et al., 2018) [35] , (Zhong et al., 2017)

Mg3LysM

Attaches to chitin, postpones the defensive reaction (Lee et al., 2014) [46] , (Marshall et al., 2011) [55]

Mg1LysM

Attaches to chitin against chitinase, but no obvious role in avoiding recognition (Lee et al., 2014) [46] , (Marshall et al., 2011) [55]

Conclusion

Host-pathogen interactions are primarily understood through biotrophic systems, where fungal effectors manipulate plant defense responses to their advantage. Necrotrophic pathogens like Z. tritici, P. tritici-repentis, and P. nodorum, while distinct, share strategies to induce host susceptibility and colonization. Z. tritici notably remains unrecognized during early colonization, unlike its counterparts. Gene-forgene recognition is observed in Z. tritici-wheat interactions, unlike in P. tritici-repentis and P. nodorum. Once the necrotrophic transition occurs, all three pathogens induce similar responses, triggering programmed cell death (PCD) and modulating antimicrobial components to obtain nutrients. Despite our understanding, gaps persist in how these pathogens evade host defenses, highlighting the need for comprehensive strategies to develop durable resistance.

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