An effector essential for virulence of necrotrophic fungi targets plant hirs to inhibit host immunity

ABSTRACT Phytopathogens often secrete effectors to enhance their infection of plants. In the case of _Sclerotinia sclerotiorum_, a necrotrophic phytopathogen, a secreted protein named

SsPEIE1 (_Sclerotinia sclerotiorum_ Plant Early Immunosuppressive Effector 1) plays a crucial role in its virulence. During the early stages of infection, _SsPEIE1_ is significantly

up-regulated. Additionally, transgenic plants expressing _SsPEIE1_ exhibit increased susceptibility to different phytopathogens. Further investigations revealed that SsPEIE1 interacts with a

plasma membrane protein known as hypersensitive induced reaction (HIR) that dampens immune responses. SsPEIE1 is required for _S. sclerotiorum_ virulence on wild-type _Arabidopsis_ but not

on _Arabidopsis hir4_ mutants. Moreover, _Arabidopsis hir2_ and _hir4_ mutants exhibit suppressed pathogen-associated molecular pattern-triggered reactive oxygen species (ROS) bursts and

salicylic acid (SA)-associated immune gene induction, all of which are phenocopied by the _SsPEIE1_ transgenic plants. We find that the oligomerization of AtHIR4 is essential for its role in

mediating immunity, and that SsPEIE1 inhibits its oligomerization through competitively binding to AtHIR4. Remarkably, both _Arabidopsis_ and rapeseed plants overexpress AtHIR4 display

significantly increased resistance to _S. sclerotiorum_. In summary, these results demonstrate that SsPEIE1 inhibits AtHIR4 oligomerization-mediated immune responses by interacting with the

key immune factor AtHIR4, thereby promoting _S. sclerotiorum_ infection. SIMILAR CONTENT BEING VIEWED BY OTHERS RLP23 IS REQUIRED FOR _ARABIDOPSIS_ IMMUNITY AGAINST THE GREY MOULD PATHOGEN

_BOTRYTIS CINEREA_ Article Open access 14 August 2020 A CONSERVED OOMYCETE EFFECTOR RXLR23 TRIGGERS PLANT DEFENSE RESPONSES BY TARGETING ERD15LA TO RELEASE NBNAC68 Article Open access 27

July 2024 N-HYDROXYPIPECOLIC ACID TRIGGERS SYSTEMIC ACQUIRED RESISTANCE THROUGH EXTRACELLULAR NAD(P) Article Open access 27 October 2023 INTRODUCTION Necrotrophic pathogenic fungi are widely

distributed and cause significant crop losses. Significant challenges posed by necrotrophs to crop production are expected to increase with climate change1,2. _Sclerotinia sclerotiorum_

(Lib.) de Bary, a destructive ascomycete fungus with a wide host range, can infect over 700 plant species, including economically important crops such as _Brassica napus_, _Glycine max_,

_Helianthus annuus_ L., _Beta vulgaris_ and _Lactuca sativa_3,4,5,6. _S. sclerotiorum_ causes substantial yield losses and economic damage on many crops. Additionally, this fungus is very

difficult to control due to the complexity of its disease cycle7,8. Studying the pathogenesis of _S. sclerotiorum_ and its interactions with host plants is essential for developing

innovative, environmentally friendly control strategies for Sclerotinia diseases. Plants have evolved complex and diverse receptor proteins to interact with pathogens. These receptors are

distributed in the cell membrane and cytoplasm, monitor the activity of pathogens, and generate various immune responses against pathogen invasion9,10. _S. sclerotiorum_ is also inevitably

recognized by plants through multiple pathways. For example, chitin in its cell wall is recognized by the receptor LYK5 on the cell membrane of _Arabidopsis_, transmitting immune signals

downstream via the co-receptor kinase CERK111,12,13. In addition, various secreted proteins of this necrotrophic fungus can be recognized by receptors on plant membranes, triggering

pattern-triggered immunity (PTI)14,15,16. The PTI signaling pathway rapidly transmits signals intracellularly and initiates early plant immune responses, including reactive oxygen species

(ROS) burst, mitogen-activated protein kinase (MAPK) activation and increased transcription of disease-resistant related genes to defend against pathogen invasion17,18,19. In contrast, _S.

sclerotiorum_ has long evolved a powerful arsenal to combat plants. Numerous studies have shown that the successful infection of plants by _S. sclerotiorum_ is closely linked to the

production of oxalic acid (OA) and cell wall degrading enzymes (CWDEs). OA plays multiple roles in the plant immune response, including creating a low pH environment for invasion,

sequestering calcium, impairing the oxidative burst of the plant, inducing programmed cell death similar to apoptosis, and inhibiting autophagy20,21,22,23,24,25,26,27. Moreover, this fungus

is induced by the host to secrete large amounts of CWDEs to break down the plant cell wall and digest plant cells, ultimately facilitating successful infection28,29,30. Equally important,

small secretory proteins are also involved in the pathogenesis of _S. sclerotiorum_. Although many secreted proteins are predicted for _S. sclerotiorum_, only a few have been empirically

investigated. For example, the secreted protein SsCaf1 is required for appressorium formation in the early stage of _S. sclerotiorum_ infection31. Additionally, _S. sclerotiorum_ finely

regulates plant immunity or metabolism by secreting effectors into plant cells. For example, the small secretory protein SsSSVP1 interacts with QCR8 and alters its mitochondrial

localization, thereby interfering with plant energy metabolism32. The secretory protein SsITL localizes in plant cell chloroplasts and interacts with CAS to suppress salicylic acid

pathway-related immunity, promoting fungal infection33. SsCP1 interacts with plant PR1 in the apoplast to promote _S. sclerotiorum_ infection, but it can also be recognized by plants to

trigger defense responses via the salicylate (SA) signaling pathway16. Recently, it was reported that SCP, a small cysteine-rich secreted protein of _S. sclerotiorum_, can be recognized by

_Arabidopsis_ RLP30 and trigger plant immunity34. The apoplast is also an important battleground for _S. sclerotiorum_ effectors. A recent study reported that _S. sclerotiorum_ secretes a

“rescuer” protein, SsPINE1, that binds to the PGIPs of the host to “rescue” the PGs, leading to successful infection35. These reports strongly suggest that the secretory proteins of _S.

sclerotiorum_ play crucial roles in the infection process. However, the strategies by which _S. sclerotiorum_ secretory proteins overcome plant resistance remain largely unexplored, making

it essential to investigate their functions to further understand the pathogenesis of _S. sclerotiorum_. Hypersensitive induced reaction (HIR) proteins belong to the SPFH

(stomatin/prohibitin/flotillin/HfK/C) superfamily, a diverse family of membrane proteins anchored in membrane micro-regions. These proteins contain a highly conserved SPFH structural domain

and are involved in regulating plant growth and responses to biotic and abiotic stresses36,37,38. HIR genes are up-regulated upon attack by pathogens, including bacteria, fungi, and

viruses39,40,41,42, and the accumulation of HIR proteins activates the host hypersensitive response (HR)39,43,44. In _Nicotiana benthamiana_, NbHIR3 can promote plant-based resistance

through EDS1 and salicylic acid-dependent pathways42. _Arabidopsis_ has four HIR genes (AtHIR1-4), with all AtHIR proteins enriched in membrane micro-regions of the plasma membrane,

mediating resistance to _Pseudomonas syringae_ pv. _tomato_ DC300040. It has been reported that AtHIR1 may recruit plant immune-associated proteins such as H+-ATPase 2 (AHA2) at the plasma

membrane to form a large complex in response to pathogen sensing45. Overexpression of HIR1 in _N. benthamiana_ can trigger cell death, and this ability is associated with the

self-interaction of HIR1 to form homo-oligomers46. Additionally, HIRs also positively regulate resistance to pathogens in wheat and rice41,42. These findings suggest that HIR proteins are

important immunoregulatory proteins in plants, but the mechanisms by which they regulate plant disease resistance are not yet clear. In this study, we identified a secreted protein important

for _S. sclerotiorum_ virulence and related to inhibition of early immunity in _A. thaliana_, named SsPEIE1 (_Sclerotinia sclerotiorum_ Plant Early Immunosuppressive Effector 1). Through

phenotypic analysis of _S. sclerotiorum_ mutants, transgenic and mutant _Arabidopsis_, we found that SsPEIE1 is essential for the full virulence of _S. sclerotiorum_ and that HIRs are

virulence targets of SsPEIE1. We also clarified the important role of AtHIR4 oligomerization in resistance to _S. sclerotiorum_. SsPEIE1 competitively interacts with AtHIR4 to impair its

oligomer formation, inhibiting plant disease resistance. This study contributes to our understanding of the virulence mechanisms and plant immune signaling networks in _S. sclerotiorum_,

potentially aiding in the development of solutions to plant diseases caused by this devastating pathogen. RESULTS SECRETORY PROTEIN SSPEIE1 PLAYS AN ESSENTIAL ROLE IN FULL VIRULENCE OF _S.

SCLEROTIORUM_ _SsPEIE1_ was identified from the transcriptome analysis as being substantially up-regulated during _S. sclerotiorum_ infection47. Phylogenetic analysis reveals that homologs

of SsPEIE1 are widespread among necrotrophic pathogenic fungi, including several significant plant pathogens (Fig. 1a), but there is no known functional domain within SsPEIE1. _SsPEIE1_

(https://www.broadinstitute.org/fungal-genome-initiative/sclerotinia-sclerotiorum-genome-project) encodes a secreted protein with 152 amino acids, and its N-terminal contains a predicted

signal peptide (18-aa). Meanwhile, SsPEIE1 possesses four conserved cysteine residues (C38, C45, C64, and C86) (Fig. 1b and Supplementary Fig. 2a). To investigate whether the SsPEIE1 signal

peptide exhibits normal secretory activity, we conducted experimental validation using the yeast secretion trap system48. YTK12 with pSUC2-Avr1bsp served as a positive control, while YTK12

and YTK12 with pSUC2 served as negative controls (Supplementary Fig. 2b). The transformed strains of YTK12 with pSUC2-SsPEIE1sp or pSUC2-SsPEIE1 were capable of degrading disaccharides to

produce glucose, which results in the production of insoluble red-colored triphenylformazan from 2, 3, 5-triphenyltetrazolium chloride (TTC), allowing them to grow regularly on YPRAA media

(Fig. 1c, d). We used RT-qPCR to verify the expression of _SsPEIE1_ in _S. sclerotiorum_ during infection. When inoculated onto leaves of _A. thaliana_ (Col-0), the transcript level of

_SsPEIE1_ rapidly increased peaking at a 68-fold increase at 6 h post-inoculation (hpi) (Fig. 1e). Notably, the transcript level of _SsPEIE1_ did not increase when _S. sclerotiorum_ was

grown on potato dextrose agar (PDA) medium (Fig. 1e), suggesting that the transcription of _SsPEIE1_ is specifically induced by host and this gene may play significant roles in _S.

sclerotiorum_ virulence. To further investigate the contribution of SsPEIE1 to _S. sclerotiorum_ virulence, we obtained two _SsPEIE1_ deletion mutants (_ΔSsPEIE1-1_ and _ΔSsPEIE1-2_) through

targeted gene replacement with a hygromycin resistance cassette (Supplementary Fig. 1a, b). We also generated complemented strains (_ΔSsPEIE1-1-C1_ and _ΔSsPEIE1-1-C2_) by introducing the

_SsPEIE1_ wild-type (WT) allele into the deletion mutant using the ATMT method (Supplementary Fig. 1c). The two Δ_SsPEIE1_ mutants showed similar growth rates, colony morphology, and OA

production capacity to the wildtype strain 1980 (Fig. 1f, g and Supplementary Fig. 1d), indicating that _SsPEIE1_ is not required for normal growth and does not affect OA production.

However, the Δ_SsPEIE1_ mutants caused significantly smaller disease lesions than the wildtype strain on _Arabidopsis_ leaves (Fig. 1h, i). The _ΔSsPEIE1_ mutants also showed lower levels of

relative _Sclerotinia_ biomass compared to WT strain (Fig. 1j). Additionally, when we inoculated _Brassica napus_ leaves with WT strain 1980 and the Δ_SsPEIE1_ mutants. Similarly,

Δ_SsPEIE1_ mutants also exhibited a significant reduction in virulence (Supplementary Fig. 1e, f). Importantly, the complementation strains (_ΔSsPEIE1-1-C1_ and _ΔSsPEIE1-1-C2_) showed

restored virulence, with lesion areas comparable to those of the wildtype strain (Fig. 1h–j). These results suggest that SsPEIE1 is a secreted protein required for the full virulence of _S.

sclerotiorum_. TRANSGENIC _ARABIDOPSIS_ PLANTS EXPRESSING SSPEIE1 HAVE INCREASED SUSCEPTIBILITY TO NECROTROPHIC PATHOGENS AND SUPPRESSED IMMUNE RESPONSES To investigate the roles of SsPEIE1

in plant-_Sclerotinia_ interaction, stable transgenic plants constitutively expressing _SsPEIE1_ (_35S:SsPEIE1-3×Flag_) were generated in wildtype _Arabidopsis_ (Col-0). The expression of

SsPEIE1 in _Arabidopsis_ was confirmed by western blot analysis (Supplementary Fig. 3a). _SsPEIE1_ transgenic lines (_oxSsPEIE1_−2, _oxSsPEIE1_−12, and _oxSsPEIE1_−22) exhibited smaller leaf

areas compared to the WT Col-0 plants (Supplementary Fig. 3b–d). We first investigated whether SsPEIE1 affects plant susceptibility to _S. sclerotiorum_ infection. Compared to WT, _SsPEIE1_

transgenic plants exhibited significantly larger disease lesion areas and more relative _Sclerotinia_ biomass (Fig. 2a–c). _SsPEIE1_ transgenic plants also showed higher levels of

susceptibility to another necrotrophic fungal pathogen _Botrytis cinerea_ (Fig. 2d–f). Furthermore, more dramatic increase in lesion area and relative _Sclerotinia_ biomass was observed when

Δ_SsPEIE1_ mutants were inoculated on _SsPEIE1_ transgenic plants compared to Col-0. The virulence of Δ_SsPEIE1_ mutants on _SsPEIE1_ transgenic plants increased to the level of WT _S.

sclerotiorum_ on WT Col-0 plants (Fig. 2g–i). These results further suggest that SsPEIE1 is an indispensable virulence factor for _S. sclerotiorum_ and ectopic expression of _SsPEIE1_ in the

host can adequately restore virulence of the Δ_SsPEIE1_ mutants to the WT level. These data indicate that SsPEIE1 may be involved in the plant-pathogen interaction by interfering with the

plant immune response and reducing plant resistance. Consistent with these virulence phenotypes, _SsPEIE1_ transgenic plants exhibited a greatly reduced MAPK activation triggered by the

PAMPs chitin or flg22 (Fig. 2j, k). Additionally, the ROS burst triggered by chitin, flg22, and _Sclerotinia_ nlp20 was significantly compromised in _SsPEIE1_ transgenic plants (Fig. 2l–n).

Furthermore, the immune marker genes _FRK1, NHL10_, and _PR1_ showed lower induction levels in _SsPEIE1_ transgenic plants upon chitin treatment (Fig. 2o). In summary, the plant early immune

response in _SsPEIE1_ transgenic plants was strongly suppressed, further highlighting the importance of SsPEIE1 in the virulence of _S. sclerotiorum_. SSPEIE1 INTERACTS WITH _ARABIDOPSIS_

HYPERSENSITIVE INDUCED REACTION 4 (ATHIR4) IN THE PLASMA MEMBRANE Since SsPEIE1 is a secreted protein and inhibits the immune response in plants, we hypothesized that it might function as a

fungal effector. We conducted a yeast two-hybrid (Y2H) screen of the _Arabidopsis_ cDNA library. The cDNA screening identified a total of 106 positive clones. Sequencing these clones

revealed 72 prey proteins (Supplementary Data 1). We validated the genes encoding proteins with a high number of clones and found that only six prey proteins interacted with SsPEIE1 (top six

in Supplementary Data 1). Since SsPEIE1-GFP localizes to the cell membrane and cytoplasm in _N. benthamiana_ (Fig. 3a), we focused on prey proteins with similar subcellular localization

that also interacted with SsPEIE1. _A. thaliana_ hypersensitive induced reaction 4 (AtHIR4) met both criteria (Supplementary Data 1 and Supplementary Fig. 5a). Targeted Y2H assays revealed a

well-defined interaction between AtHIR4 and SsPEIE1 in yeast (Fig. 3b). We also performed a split-luciferase (split-LUC) assay to investigate the association of AtHIR4 with SsPEIE1. Results

indicated that AtHIR4 fused with the C-terminal LUC produced a strong LUC signal when co-expressed with SsPEIE1-nLUC in _N. benthamiana_ leaves, with similar results observed when the

positions of AtHIR4 and SsPEIE1 were switched (Fig. 3c). These results were further confirmed by Co-IP assay in _N. benthamiana_ leaves showing that GFP or FLAG-tagged SsPEIE1 could

co-immunoprecipitate with FLAG or GFP-tagged AtHIR4 (Fig. 3d). Co-expression of SsPEIE1 and AtHIR4 in _Arabidopsis_ protoplasts yielded similar results to those obtained from Co-IP

experiments in _N. benthamiana_ (Supplementary Fig. 5b). When SsPEIE1-mCherry and AtHIR4-GFP were co-expressed in _N. benthamiana_ leaves, the mCherry and GFP signals overlapped perfectly,

indicating that SsPEIE1 and AtHIR4 co-localize in the plasma membrane (Fig. 3e). These data demonstrate that SsPEIE1 physically interacts with AtHIR4 in plants. ATHIR2 AND ATHIR4 PLAY

ESSENTIAL ROLES IN RESISTANCE TO NECROTROPHIC FUNGI Previous research has shown that HIR proteins are located on the cell membrane and play a positive regulatory role in tomato resistance to

Tomato leaf curl Yunnan virus (TLCYnV)46. To explore the role of AtHIR4 in regulating plant immune responses to _S. sclerotiorum_, we obtained a T-DNA insertion mutant _Arabidopsis_ line,

_hir4_, from Arashare and bred it to obtain a purified mutant (Supplementary Fig. 3e). Loss of function of AtHIR4 in _Arabidopsis_ did not affect on plant morphology or growth (Supplementary

Fig. 3f). However, the _hir4_ mutant exhibited significantly increased susceptibility to _S. sclerotiorum_ and _B. cinerea_ (Fig. 4a, d), with lesion areas and relative fungal biomass

increased by ~50% (Fig. 4b, c, e, f), similar to the _SsPEIE1_ transgenic plants. Both the _hir4_ mutant and _SsPEIE1_ transgenic plants also showed impaired chitin-induced expression of

early immune marker genes (Supplementary Fig. 6a). Additionally, the _hir4_ mutant and _SsPEIE1_ transgenic plants exhibited defects in resistance to the foliar hemi-biotrophic bacterial

pathogen _Pst_ DC3000 (Supplementary Fig. 6b, c). These data indicate that AtHIR4 is indispensable for plant resistance to both fungal and bacterial pathogens. Notably, the virulence of

_ΔSsPEIE1_ mutant strains on _hir4_ mutant plants was restored (Fig. 4g–i). These results further suggested that SsPEIE1 inhibits the plant immune response by interacting with AtHIR4. In

addition to AtHIR4, _A. thaliana_ contains three other AtHIR proteins: AtHIR1, AtHIR2, and AtHIR3. To determine whether SsPEIE1 interacts with any of the other HIRs, we performed Y2H and

split luciferase assays. The results showed that SsPEIE1 also associates with AtHIR1, AtHIR2, and AtHIR3 (Supplementary Fig. 7a, b). The expression levels of the HIRs are rapidly induced by

biotic stresses40,46. We examined the transcription patterns of _AtHIR_ genes in _Arabidopsis_ during _S. sclerotiorum_ infection. The results indicated that the expression levels of

_AtHIRs_ significantly increased at the early stages of _S. sclerotiorum_ infection (1 hpi), followed by a gradual decrease in _AtHIR1_ and _AtHIR3_ expression. In contrast, AtHIR2 and

AtHIR4 expression levels decreased at 3 hpi and then significantly re-increased at 12 hpi (Fig. 4j). These results suggest that _AtHIR_ genes are significantly induced in the early stages of

_S. sclerotiorum_ infection, with AtHIR2 and AtHIR4 possibly involved in the later immune response to resist _S. sclerotiorum_ invasion in _Arabidopsis_. To further clarify the biological

significance of AtHIR2 and AtHIR4 in plant immunity, we edited _AtHIR2_ in _hir4_ mutant _Arabidopsis_ to obtain the _hir2/hir4_ double mutant _Arabidopsis_ (_hir24_) (Supplementary Fig. 

3f–h). We then separately challenged _hir2_, _hir4_, and _hir24_ mutants with _S. sclerotiorum_. The _hir2_, _hir4_, and _hir24_ mutants exhibited faster disease development and higher

biomass of _S. sclerotiorum_ than the WT plants Col-0 (Fig. 4k–m), highlighting the important roles of AtHIR2 and AtHIR4 in regulating resistance to _S. sclerotiorum_. Additionally, the

virulence of the _ΔSsPEIE1_ mutant strains on _hir24_ mutant plants was restored to the level of the WT strain (Fig. 4n–p). Consistent with the phenotypes of _SsPEIE1_ transgenic plants, the

_hir2_, _hir4_, and _hir24_ mutants also exhibited impaired chitin-triggered MAPK activation and ROS burst, especially in the _hir24_ double mutant _Arabidopsis_ (Fig. 4q, r). Furthermore,

chitin-induced up-regulation of early immune marker genes was significantly reduced in _hir2_ or _hir4_ mutant lines (Supplementary Fig. 6d). These results support that both AtHIR2 and

AtHIR4 are important for early plant immune responses and further clarify that the plant susceptibility induced by SsPEIE1 is achieved through its targeting of AtHIR2 and AtHIR4. SSPEIE1

COMPETITIVELY BINDS TO ATHIR4 AND DISRUPTS ITS OLIGOMERIZATION CAPACITY AND OLIGOMERIZATION-MEDIATED DISEASE RESISTANCE HIR proteins are indispensable for plant immunity, and recent studies

have shown that interfering with the homo-oligomerization of HIR proteins significantly affects both HIR-mediated HR and disease resistance46,49. We found that AtHIR4 can form homo-oligomers

in the Co-IP experiments, but this homo-oligomer formation is obviously reduced in the presence of SsPEIE1 (Fig. 3d). The same phenomenon was also observed when AtHIR4 and SsPEIE1 were

co-expressed in _N. benthamiana_ (Fig. 5a). These observations led us to hypothesize that AtHIR4 undergoes homo-oligomerization and that SsPEIE1 can interact with AtHIR4 to impair this

process. To further confirm this hypothesis, we performed both in vivo and in vitro validation experiments. First, the yeast three-hybrid (Y3H) assay showed that SsPEIE1 expression was

repressed in the presence of methionine (Met), allowing yeast strains to grow on SD/-Leu/-Trp/-His medium, suggesting that AtHIR4 can self-interact. However, in the absence of Met, SsPEIE1

transcription was activated, and strains expressing SsPEIE1 failed to grow normally on SD/-Leu/-Trp/-His-Met medium, indicating that SsPEIE1 significantly inhibits AtHIR4 self-interaction

(Fig. 5b). To determine whether SsPEIE1 directly affects AtHIR4 self-interactions, we conducted in vitro pull-down assays using MBP, MBP-SsPEIE1, GST-AtHIR4, and AtHIR4-His fusion

recombinant proteins. MBP-SsPEIE1 was pulled down by GST-AtHIR4, and as the amount of MBP-SsPEIE1 increased, the amount of AtHIR4-His pulled down by GST-AtHIR4 gradually reduced. This not

only indicated a direct interaction between AtHIR4 and SsPEIE1 in vitro but also confirmed that SsPEIE1 inhibits the self-association of AtHIR4 in a dose-dependent manner (Fig. 5c).

Additionally, we performed Co-IP assays where AtHIRs-Flag, AtHIRs-Myc, and SsPEIE1-GFP or GFP were co-expressed in _N. benthamiana_ leaves. The results showed that AtHIRs-Myc could be

strongly immunoprecipitated with AtHIRs-Flag, confirming that AtHIR4 can self-interact to form homo-oligomers. However, the interaction between AtHIRs-Flag and AtHIRs-Myc was significantly

weakened in the presence of SsPEIE1 (Fig. 5d). These findings indicate that SsPEIE1 competitively binds to AtHIR4, impairing its self-interaction. To further clarify the role of AtHIR4

oligomerization in plant resistance to _S. sclerotiorum_ and the biological significance of SsPEIE1 impairing AtHIR4 self-interaction, we predicted the key amino acid site for AtHIR4

homo-oligomer formation using Alphafold 2. Mutating the valine at position 157 to glycine (AtHIR4v157a) significantly reduced its oligomer formation ability (Supplementary Fig. 8b and

Supplementary Fig. 9b, c). AtHIR4v157a exhibited reduced self-association compared to wildtype AtHIR4 in Co-IP experiments (Fig. 5e). Through Y2H assays, we confirmed that the valine at

position 157 of AtHIR4, as well as homologous valines in other HIR family members, are essential for their self-interactions (Supplementary Fig. 9d). Additionally, the interaction between

SsPEIE1 and AtHIR4v157a was weakened (Fig. 5f). To elucidate the effect of AtHIR4 oligomerization on plant disease resistance, GFP, SsPEIE1, AtHIR4v157a, and AtHIR4 were expressed in _N.

benthamiana_ leaves and inoculated with _S. sclerotiorum_ 36 h after infiltration (Fig. 5h and Supplementary Fig. 8b). The results showed that _SsPEIE1_ expression made _N. benthamiana_ more

susceptible to _S. sclerotiorum_, consistent with the inoculation of _SsPEIE1_ transgenic _Arabidopsis_ (Fig. 2a). Expression of AtHIR4 significantly increased the resistance of _N.

benthamiana_ to _S. sclerotiorum_, but AtHIR4v157a lost the ability to enhance the plant resistance (Fig. 5g). The lesion area on AtHIR4v157a expressing leaves did not differ significantly

from that on GFP-expressing leaves (Fig. 5i). The relative _Sclerotinia_ biomass also showed a more consistent trend (Fig. 5j). These results illustrate that AtHIR4 oligomer formation is

crucial for plant resistance to _S. sclerotiorum_, while SsPEIE1 promotes plant susceptibility by interacting with AtHIR4 to inhibit its homo-oligomer formation. CONSTITUTIVE OVER-EXPRESSION

OF _ATHIR4_ ENHANCES PLANT RESISTANCE AtHIR4 is indispensable for plant immunity, and its absence leads to increased susceptibility to _S. sclerotiorum_. We further investigated the active

role of AtHIR4 in plant disease resistance as a potential method for developing disease-resistant plants. To rapidly assess the function of AtHIR4, we generated transgenic _Arabidopsis_

plants constitutively overexpressing AtHIR4 (Supplementary Fig. 10a). The _35S:AtHIR4-3×Flag Arabidopsis_ plants exhibited slightly smaller aboveground morphology and greener leaves compared

with wildtype Col-0 at 4 weeks of growth (Supplementary Fig. 10b). At the same time, _AtHIR4_ overexpression significantly enhanced plant resistance to both _S. sclerotiorum_ and _B.

cinerea_ (Fig. 6a–f). We also examined the intensity of the early immune response by treating _AtHIR4_-overexpressing _Arabidopsis_ with chitin. The results showed that

_AtHIR4_-overexpressing plants exhibited stronger ROS burst and activation of MAPKs compared to WT plants (Fig. 6g, h). More significantly, we also created transgenic rapeseed plants

expressing _AtHIR4_ by infecting hypocotyls with _A. tumefaciens_50 (Supplementary Fig. 10c). The growth phenotype of transgenic rapeseed was not noticeably different from that of WT plants

(XiaoYun, Y127)51 and empty vector plants (Supplementary Fig. 10d and Supplementary Table 2). Consistent with _AtHIR4_-overexpressing _Arabidopsis_ plants, transgenic rapeseed plants

expressing AtHIR4 showed marked increase in resistance to _S. sclerotiorum_ and _B. cinerea_ (Fig. 6i–n). These results further indicate that AtHIR4 does play an important role in plant

resistance and that its function is conserved among different plant species. DISCUSSION As a necrotrophic phytopathogen, _S. sclerotiorum_ possesses powerful weapons, including a large array

of CWDEs and OA secretion, which contribute to its wide host range and strong virulence. While the mechanisms underlying plant resistance to biotrophic pathogens have been extensively

studied, much less is known about the resistance mechanisms against necrotrophs52. Recent studies have predicted that numerous secreted proteins are involved in _S. sclerotiorum_

infection5,53,54, and are crucial for its virulence. Some secreted proteins of _S. sclerotiorum_ have been experimentally identified and characterized, with most reported as elicitors

associated with cell death55,56,57. However, only a few effectors have been studied in depth. In this study, we demonstrated that the plant membrane-localized SsPEIE1, a core effector of _S.

sclerotiorum_, targets multiple host HIR factors to suppress immunity. Notably, SsPEIE1 disrupts the integrity of the plant immune system by impairing the oligomerization of AtHIR4, leading

to compromised host resistance (Fig. 5). Specifically, this fungal effector weakens critical immune responses of the host (Fig. 2). We found that the virulence of _ΔSsPEIE1_ mutants was

significantly reduced but restored in SsPEIE1-complemented strains, confirming that SsPEIE1 is essential for the full virulence of _S. sclerotiorum_ (Fig. 1h). Furthermore, the expression of

SsPEIE1 in _Arabidopsis_ restored the lost virulence of _ΔSsPEIE1_ mutants, further substantiating the key role of SsPEIE1 in manipulating host defense and facilitating pathogen infection.

Additionally, a homologous protein of SsPEIE1 is present in many necrotrophic fungal pathogens, including _B. cinerea_. Importantly, our inoculation results revealed that SsPEIE1 transgenic

_Arabidopsis_ were significantly more susceptible to _B. cinerea_ (Fig. 2d–f) than the WT Col-0, indicating that PEIE1 is a conserved effector in necrotrophic pathogenic fungi. Previous

research on the secreted proteins of _S. sclerotiorum_ has largely focused on their role in inducing plant cell death, which can facilitate _S. sclerotiorum_ colonization16,32,57. However,

early plant immunity is also crucial for _S. sclerotiorum_ pathogenesis, with ROS bursts in plants negatively affecting early invading _S. sclerotiorum_58,59. We discovered that SsPEIE1

interacts with AtHIR4 in plants, significantly inhibiting early immune responses to chitin and flg22 in _Arabidopsis_ expressing SsPEIE1 (Figs. 2j–n and 3). This provides direct evidence

that the _S. sclerotiorum_ effector inhibits early immunity in plants. It was proposed that _S. sclerotiorum_ may have a transient biotrophic phase during infection and secrete effectors to

suppress plant immunity60. Indeed, _S. sclerotiorum_ likely controls the onset of plant cell death at specific infection stages, thereby negatively impacting plant immunity beforehand61.

Notably, _hir4_ mutants exhibited similar immunosuppression and susceptibility to necrotrophic fungal pathogens as _SsPEIE1_ transgenic _Arabidopsis_ (Fig. 4a–f and Supplementary Fig. 6a),

and the virulence of Δ_SsPEIE1_ mutants was obviously restored on _hir4_ and _hir24_ mutant _Arabidopsis_ plants (Fig. 4g, h, i, n, o, p). Additionally, _hir4_ mutant was less responsive to

flg22 and more susceptible to _Pst_ DC3000, consistent with the phenotype of _SsPEIE1_ transgenic _Arabidopsis_ (Supplementary Fig. 6b, c and Supplementary Fig. 11c, d). These findings

provide strong genetic evidence that AtHIR4 is targeted by SsPEIE1 as a crucial positive regulator of plant immunity. HIR proteins have been extensively studied as positive regulators of

plant HR in _Solanaceae_39,62. HIR proteins in the _Solanaceae_ family induce HR in _N. benthamiana_, and the expression of CaHIR1 and OsHIR1 in _Arabidopsis_ significantly enhances

resistance, although excessive immune responses can lead to _Arabidopsis_ dwarfing43,44. In contrast, reduced HR and increased accumulation of PVX-CP protein were observed in _N.

benthamiana_ silencing _HIR1_46. Current studies suggest that HIR proteins likely play active roles in virus and bacterial infections through the regulation of the salicylic acid

pathway40,42,46. However, the role of HIR in fungal resistance remains unclear. We found that the transcripts of _AtHIR2_ and _AtHIR4_ were significantly up-regulated during _S.

sclerotiorum_ infection (Fig. 4j), suggesting that AtHIR2 and AtHIR4 might play crucial roles in resistance to _S. sclerotiorum_. We further evaluated the effects of AtHIR2 and AtHIR4 on

Arabidopsis resistance, finding that hir2, hir4, and hir24 mutants were more susceptible to various pathogens, particularly _S. sclerotiorum_. (Fig. 4k–m and Supplementary Fig. 11a, b). Our

study reveals that AtHIR2 and AtHIR4 in _Brassicaceae_ are indispensable for resistance to fungal pathogens. Furthermore, overexpression of AtHIR4 in _Arabidopsis_ resulted in high levels of

resistance to necrotrophic fungi and the bacterial pathogen _Pst_ DC3000 (Supplementary Fig. 11e, f). Concurrently, _hir2_, _hir4_, and _hir24_ mutants exhibited reduced early immune

responses to treatments with PAMPs from different pathogen sources (Fig. 4q, r and Supplementary Fig. 11c, d), whereas _Arabidopsis_ overexpressing AtHIR4 showed significantly enhanced early

immune responses (Fig. 6g, h and Supplementary Fig. 11g, h). These biochemical changes in resistance are consistent with the genetic experiment results. Additionally, HIR1 has been reported

to play a role in antiviral processes46. The related results provide ample evidence that HIR proteins are broad-spectrum and vital plant disease resistance proteins, playing critical roles

in resistance to a wide range of pathogens. To elucidate the significance of the interaction between SsPEIE1 and HIR proteins, we co-expressed SsPEIE1 and AtHIR4 both in vivo and in vitro

and found that SsPEIE1 inhibits the oligomerization of AtHIR4 by competitively binding to it (Fig. 5a–d). Previous studies have shown that MdHIR can self-interact to form homo-oligomers,

which is essential for its pathogen resistance capability49. Similarly, the TLCYnV C4 protein interacts with HIR1 and promotes the interaction of NbLRR1 with HIR1, thereby interfering with

HIR1 oligomerization and leading to HIR1 degradation46. Our study further clarified the correlation between AtHIR4 oligomerization and resistance, demonstrating that impaired homo-oligomer

formation of the AtHIR4v157a variant significantly reduces disease resistance (Fig. 5e, g, i, j). These findings elucidate the interaction mechanism between SsPEIE1 and AtHIRs, highlighting

that AtHIR4 oligomerization is essential for resistance. Additionally, HIR proteins are widely present in various _S. sclerotiorum_ hosts, and we verified that SsPEIE1 can interact with

CaHIR1 and SlHIR1 in _Solanaceae_ plants (Supplementary Fig. 7a). This suggests that SsPEIE1 likely employs a similar mechanism to inhibit the immune function of HIR proteins in other hosts,

consistent with the broad host range of _S. sclerotiorum_. The defense-related hormone salicylic acid (SA) plays significant roles in local and systemic resistance in plants63, activating

specific immune pathways and the expression of thousands of genes, including key components of plant resistance64. Our results demonstrated that the up-regulated expression of chitin-induced

SA pathway-related gene was significantly suppressed in _hir2_, _hir4_ and _SsPEIE1_ transgenic _Arabidopsis_ (Fig. 2o and Supplementary Fig. 6d). Furthermore, we found that chitin-induced

MAPKs phosphorylation and ROS bust were negatively affected in _hir2_, _hir4_, and _hir24_ mutant _Arabidopsis_ (Fig. 4q, r), consistent with the phenotype of SsPEIE1 transgenic

_Arabidopsis_ (Fig. 2j, l). These immune responses are critical for SA pathway-mediated plant resistance and SA has been reported to play an active role in resistance to _S.

sclerotiorum_19,33. Importantly, both _Arabidopsis_ and rapeseed overexpressing AtHIR4 showed enhanced resistance to _S. sclerotiorum_ and _B. cinerea_ (Fig. 6a, d, i, l). Our study

uncovered the critical roles of AtHIR2 and AtHIR4 in _Arabidopsis_ resistance to fungal pathogens, indicating that their resistance function likely depends on the SA pathway. HIR-activated

immune responses are closely related to SA, and NbHIR3-mediated HR requires EDS1 for SA response42. Elevated PR1 transcripts were observed in _oxCaHIR1_ and _oxOsHIR1_ transgenic

_Arabidopsis_, resistance to _Pst_ DC3000 was significantly increased39,44. Therefore, we hypothesize that overexpression of _AtHIR4_ enhances the immune response through the SA pathway. Our

current understanding of HIRs is limited, and HIR-mediated plant disease resistance may involve complex pathways, potentially including the jasmonic acid or ethylene pathways. However, we

have not yet identified key proteins interacting with HIRs in these pathways, and the mechanism of HIR-mediated disease resistance requires further exploration. Overall, our studies

comprehensively illustrate the critical role of SsPEIE1 as a virulence factor of _S. sclerotiorum_. We found that SsPEIE1 disrupts the oligomerization of AtHIR4 by competitively binding to

it, thereby inhibiting plant immunity. Consequently, SsPEIE1 creates a favorable environment within hostile plant for early infection and successful colonization by _S. sclerotiorum_. The

enhanced resistance to _S. sclerotiorum_ exhibited by _oxAtHIR4_ transgenic rapeseed also highlights the potential value of HIR proteins for further investigation. This unique interaction

mode between necrotrophic pathogenic fungi and plants offers fresh insights into the dynamic relationship between necrotrophic pathogens and their host plants. METHODS FUNGAL AND BACTERIAL

STRAINS, PLANT MATERIALS, AND GROWTH CONDITIONS The _S. sclerotiorum_ WT strain 1980 was cultured on PDA plates at 20 °C and stored on PDA at 4 °C. Gene deletion mutants and their

complementation mutants were cultured on PDA plates amended with 100 μg · mL−1 hygromycin B or 100 μg · mL−1 G418 (Sigma-Aldrich) (Supplementary Table 2). _Pseudomonas syringae_ pv. _tomato_

strain _Pst_ DC3000 was grown in King’s B (KB) medium containing 50 mg · mL−1 rifampicin at 28 °C. _Agrobacterium tumefaciens_ strain GV3101 carrying different constructs was incubated in

LB medium with 50 mg · mL−1 rifampicin and respective antibiotics at 28 °C. All _A. thaliana_ plants used in this study were of the Columbia-0 (Col-0) genetic background. _Arabidopsis hir1_,

_hir2, hir3_, and _hir4_ mutants were obtained from AraShare (https://www.arashare.cn/index/). _Arabidopsiss_ lines were grown in growth chamber soil at 22 °C, 75 mE−2 · s−1 (T5 LED tube

light, 4000 K). The light/dark photoperiod was 12 h and relative humidity was 40%–60%. _N. benthamiana_ plants were grown in jiffy pots in a growth chamber, under the same conditions as

described above. Leaves of 4-week-old _N. benthamiana_ plants were used for _Agrobacterium_-mediated transient expression (Supplementary Table 2). PROTEIN BIOINFORMATICS ANALYSIS The SsPEIE1

protein sequence was downloaded from the NCBI GenBank database. SIGNALP 4.0 and SIGNALP 4.1 were used for signal peptide prediction65,66, DeepTMHMM (https://dtu.biolib.com/DeepTMHMM) was

used for prediction of trans-membrane helices. Protein homology modeling was performed under the AlphaFold2 server with normal modeling mode67, and the PDB file produced was edited by PYMOL

software. Sequence alignment was performed in the genome database using the BLASTP program to obtain homologous sequences of SsPEIE1 in other species. Multiple comparisons of amino acid

sequences were generated by the DNAMAN program. Phylogenetic analysis was performed to reconstruct the phylogenetic tree using MEGA X using the maximum likelihood method. PLASMID

CONSTRUCTION AND GENERATION OF TRANSGENIC PLANTS The coding sequence of SsPEIE1 was amplified from _S. sclerotiorum_ cDNA using primers containing homologous fragments of the pCNF3 vector.

The PCR-amplified fragment was then cloned into CaMV 35S promoter-driven binary expression vectors pCNF3 with 3 × FLAG and pTF101 with GFP tags fused at the C terminus for assays in _N.

benthamiana_ and _Agrobacterium_-mediated floral dipping of _Arabidopsis_. The coding sequence of _AtHIR_ genes was amplified from _Arabidopsis_ Col-0 cDNA and cloned into the pCNF3 vector

for expression in _N. benthamiana_ and _Agrobacterium_-mediated floral dipping of _Arabidopsis_, and _AtHIR_ genes were subcloned into a binary expression vector driven by the CaMV 35S

promoter. The AtHIR4 mutant AtHIR4v157a was subcloned into the pCNF3, pCAMBIA 1300 (Luc) and pCAMBIA 2300 (2×myc) vectors for expression in _N. benthamiana_ using homologous recombination

(Vazyme, Cat. C113-02) (Supplementary Table 1). _Agrobacterium tumefaciens_ GV3101 carrying pCNF3-SsPEIE1-3×FLAG or pCNF3-AtHIR4-3×FLAG was cultured overnight in LB liquid medium containing

50 mg · mL−1 rifampicin and 50 mg · mL−1 kanamycin. After centrifugation at 3000 × _g_ for 5 min, the bacterial cells were suspended in _Agrobacterium_ infiltration buffer containing 5%

sucrose and 0.04% (v/v) Silwet L-77 to an optical density (OD) 600 = 0.8. _Arabidopsis_ buds were thoroughly immersed in the bacterial suspension, and the plants were kept moisturized for 8 

h after immersion. Plants were then maintained at 22 °C and 45% relative humidity with a 16 h light/8 h dark photoperiod, conditions for seed harvesting. Transgenic _Arabidopsis_ was

screened with 1/2 MS containing 50 mg · mL−1 kanamycin and further confirmed by immunoblotting using anti-FLAG antibody (Sigma-Aldrich F1804). GENE KNOCKOUT AND COMPLEMENTATION OF _S.

SCLEROTIORUM_ Gene knockout mutants of the _SsPEIE1_ gene in _S. sclerotiorum_ were generated using the split-marker technique68. The knockout strategy is illustrated in Supplementary Fig. 

1a. Two fragments of ~1000 bp each, _SsPEIE1_−5′ and _SsPEIE1_−3′, flanking the gene were amplified from genomic DNA by PCR reactions with primers P1/P2 (both containing SalI sites) and

P3/P4 (both containing XbaI sites) using KOD DNA Polymerase (TOYOBO) (Supplementary Data 2). These PCR products were cloned into the _Sal_ I and XbaI sites in the pUCH18 vector containing

the hygromycin-resistant cassette, respectively16. The pUCH18-_SsPEIE1_−5′ and pUCH18-_SsPEIE1_−3′ constructs were obtained after successful cloning (Supplementary Table 1). The fusion

sequences _SsPEIE1_−5′ and _SsPEIE1_−3′ with two truncated hygromycin-resistant genes, _SsPEIE1_−5′-HY and YG-_SsPEIE1_−3′, were mass amplified with primers P1/HY and YG/P4, respectively

(Supplementary Data 2). The purified _SsPEIE1_−5′-HY and YG-_SsPEIE1_−3′ DNA fragments (10 μg or more each) were mixed in equimolar amounts and used for transformation to generate _SsPEIE1_

knockout mutants. To obtain complementary strains, we amplified the full length of the SsPEIE1 gene, including its promoter sequence, from WT genomic DNA to obtain a 5′-_Xho_I- promoter

_SsPEIE1_-_SsPEIE1_-_Kpn_I-3′ fragment, which was then cloned into the pCETNS vector containing the geneticin resistance expression cassette (Supplementary Data 2 and Supplementary Table 1).

The promoter _SsPEIE1_-_SsPEIE1_ fragment fused with the geneticin resistance expression cassette was subsequently amplified from the vector using KOD FX DNA Polymerase (TOYOBO), purified

and used in at least 10 μg quantities to transform the protoplasts of the _SsPEIE1-_knockout strain. For the preparation and transformation of _S. sclerotiorum_ protoplasts, mycelial balls

grown in PDB for 36 h were lysed with lysing enzymes (10 mg/mL) from _Trichoderma harzianum_ (Sigma Cat. L1412) to obtain fresh protoplasts16. Fragments of _SsPEIE1_−5′-HY and

YG-_SsPEIE1_−3′ were transferred into the protoplasts of WT strains covered with RM medium containing 200 μg · mL−1 hygromycin B. Putative transformants with hygromycin B resistance were

obtained after 5–7 days. After verifying the correct substitution site according to the knockout strategy schematic, the transformants were induced to produce sexual state ascospores, which

were then screened for hygromycin resistance to obtain pure syngeneic knockout mutant strains. After obtaining a pure _SsPEIE1_ gene knockout mutant and transferring the

promoter_SsPEIE1_-_SsPEIE1_-PtrpC-_Npt_II-TtrpC fragments into its protoplast, complemented transformant strains were obtained by geneticin resistance screening of the recovered mycelium.

Subsequently, genomic DNA and total RNA were extracted from the putative back-complemented transformants, and reverse transcription of the RNA was performed to obtain cDNA. The DNA and cDNA

of the putative transformants were amplified by PCR to verify the presence of the _SsPEIE1_ gene in the positive transformants (Supplementary Data 2). Gel electrophoresis, vector cloning,

and sequencing were performed using standard procedures69. DETERMINATION OF THE BIOLOGICAL CHARACTERISTICS OF _S. SCLEROTIORUM_ TRANSFORMANTS Transformant strains of _S. sclerotiorum_

(_ΔSsPEIE1_ mutant and complementary strains) were characterized for growth rate, colony morphology, acid production capacity, and virulence. The WT, knockout, and complementary strains were

inoculated in the center of PDA plates for 3 days at 20 °C to measure the growth rate and were incubated continuously for 14 days to record their colony morphology by photograph. To assay

the ability of OA production, the WT strain, deletion mutants, and back-complemented strains were inoculated into the center of PDA plates containing 0.005% (w/v) bromophenol blue dye and

grown at 20 °C for 36 h. Their acid production ability was qualitatively characterized by visual observation. FUNGAL AND BACTERIAL INOCULATION ASSAY For the fungal inoculation assay, agar

discs (2 mm in diameter) were punched from the actively growing edge of fungal 2 × SY plates (SY medium: 0.5% (w/v) sucrose and yeast extract, 1% (w/v) agar and inoculated onto the leaves of

4–5-week-old _Arabidopsis_ plants, which were then incubated at 22 °C. Six to twelve biological replicates per treatment, taken from six _Arabidopsis_ plants. Fungal virulence was assessed

macroscopically by measuring the long and short axes of the lesion with a caliper, and molecularly by measuring the ratio of _S. sclerotiorum_ DNA to host plant DNA in infected leaves.

Disease lesion area was calculated based on the elliptical area formula. Relative fungal pathogen biomass was determined by the ratio of pathogen DNA to host plant DNA70. In each group of

_Arabidopsis_ leaves, after measuring the lesion area, equal area samples were taken from the infected sites using a 1.5-cm-diameter punch (or in the case of rapeseed leaves, with a 2.5-cm

side square). The DNA of the samples was extracted and analyzed for the relative content of fungal pathogens and plant DNA by qPCR. Each treatment included three replicates (each replicate

contained 2–4 diseased leaves), and each experiment was performed three times. Primers used are provided in Supplementary Data 2. For bacterial inoculation assay, _Pst_ DC3000 was grown

overnight in KB medium supplemented with the appropriate antibiotics. After centrifugation, the bacterial cells were resuspended with 10 mM MgCl2 to a desired density. Leaves of 4-week-old

_Arabidopsis_ plants were soaked in the bacterial suspension and 2 days later the leaves were collected to detect the bacterial population. 12–24 leaves were divided into 6–12 replicates and

then ground in 100 μL of 10 mM MgCl2 and serially diluted onto TSA medium containing the appropriate antibiotics. Colony-forming units (CFUs) were counted after 3 days of incubation at 28 

°C. ROS PRODUCTION ANALYSIS AND MAPK ACTIVATION ASSAY The third or fourth pair of true leaves from 4-week-old soil-grown _Arabidopsis_ plants were cut into leaf discs (5 mm in diameter) and

further cut into strips. The leaf strips were floated in 96-well plates with 100 µL ddH2O and shaken gently overnight to eliminate the wounding effect. For the assay, ddH2O was replaced with

100 µL reaction solution containing 50 µM L-012 (Wako CAS:143556-24-5), 10 µg · mL−1 horseradish peroxidase (Sigma Cat. P6782), and appropriate elicitors (10 μg · mL−1 chitin, 1 μM Ssnlp20

or 100 nM flg22). Measurements were taken immediately after the addition of the reaction solution using a Multimode Reader Platform (Tecan Austria GmbH, SPARK 10 M), with ROS values

representing the relative light units of different plants. _Arabidopsis_ seedlings grown on 1/2 MS plates for 10 days were transferred to 1 mL sterilized ddH2O, allowed to recover overnight,

and then treated with the indicated concentrations of flg22 or chitin for 0, 5, 15, and 30 min. Total protein from the samples was extracted using protein extraction buffer (20 mM Tris-HCl,

pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, and 1% Triton X-100), and the samples were incubated at 95 °C for 10 min. The supernatant was collected after centrifugation at 10,000 × _g_

for 2 min, and the protein samples containing 1 × SDS buffer were loaded onto 10% (v/v) SDS-PAGE gels and immunoblotted with anti-PERK1/2 antibody to detect pMPK3, pMPK4, and pMPK6 (CST Cat.

9101S). SECRETION TRAP SCREEN ASSAY In this study, the predicted signal peptide fragment of the _SsPEIE1_ and _SsPEIE1__sp_ gene was fused to the N-terminus of the secretion-defective

invertase gene (suc2) in the vector pSUC2 and then transformed into the yeast strain YTK12. Candidate yeast transformants were screened and cultured on medium lacking tryptophan (CMD-W) and

YPRAA (10 g · L−1 yeast extract, 20 g · L−1 peptone, 20 g · L−1 raffinose, 2 mg · L−1 antimycin A, and 2% agar) medium, and strains with secretion activity were able to grow on YPRAA. TTC

was used to assay the secretion sucrase activity of the candidate yeast transformants. The candidate yeast transformants were incubated in 10% sucrose solution at 30 °C for 35 min, then the

supernatant was centrifuged and the final concentration of 0.1% TTC reagent was added and left at room temperature for 5 min to observe the color change in the test tube. A positive reaction

changed from colorless to dark red, with the _Avr1b__sp_ transformant, YTK12-pUSC2, and YTK12 strains used as positive and negative controls, respectively. YEAST TWO-HYBRID AND THREE-HYBRID

ASSAYS For the Y2H assay, the coding sequences of the genes to be tested for interaction (without signal peptides) were PCR amplified and cloned into pGBKT7 and pGADT7 to generate bait and

prey vectors, respectively, based on the Matchmaker Gold Yeast Two-Hybrid System for GAL4. The bait and prey plasmids were co-transformed into yeast strain Y2H Gold according to the

manufacturer’s instructions. Transformed yeast cells were grown on synthetic dropout (SD)/-Trp-Leu plates for 3–4 days and single-colony cells were transferred to 2 mL of liquid SD/-Trp-Leu

medium and cultured for 24 h. The cells were collected by centrifugation, adjusted to a concentration of 106 cells · mL−1 with sterile water, and 2 μL of yeast suspension was assayed for

growth on SD/-Trp-Leu-His-Ade plates containing 5-bromo-4-chloro-3-indolyl α-D-galactopyranoside (X-α-gal). For the Y3H assay, AtHIR4 and SsPEIE1 were cloned into the pBridge plasmid to

obtain pBridge-AtHIR4-SsPEIE1(-Met)-BD, and AtHIR4 was cloned into pGADT7 to obtain AtHIR4-AD (Supplementary Table 1). Co-expression of pBridge-AtHIR4-SsPEIE1(-Met)-BD with AtHIR4-AD was

performed in yeast strain Y2H Gold, with pBridge-AtHIR4-SsPEIE1(-Met)-BD and pGADT7 as a negative control, and pBridge-AtHIR4-MCS(-Met)-BD and AtHIR4-AD as a positive control. Yeast growth

was analyzed on SD/-Leu/-Trp/-His medium with or without methionine (Met). The pBridge plasmid contains two promoters: the yeast constitutive expression promoter ADH1 and the methionine

deficiency-inducible promoter MET25. In the absence of methionine, the yeast strain containing the pBridge-AtHIR4-SsPEIE1(-Met)-BD plasmid is induced to express SsPEIE1, while SsPEIE1

expression is repressed in the presence of methionine. Based on these principles, the effect of SsPEIE1 on AtHIR4 self-interaction was further clarified. CO-IP ASSAYS For co-IP in _N.

benthamiana_ and _Arabidopsis_ protoplasts, the cDNAs sequences of SsPEIE1 and AtHIR4 were constructed into binary expression vectors and transferred into _A. tumefaciens_ strain GV3101 by

electroporation, respectively. The proteins were co-expressed in leaves of _N. benthamiana_ by _Agrobacterium_-mediated transient expression for 36 h. Alternatively, _Arabidopsis_

protoplasts were transfected with indicated plasmids and incubated for 12 h. Subsequently, samples were then collected and lysed after vortexing in Co-IP Buffer (20 mM Tris-HCl, pH 7.5, 100 

mM NaCl, 1 mM EDTA, 2 mM DTT, 10% glycerol, 0.5% Triton X-100, and protease inhibitor cocktail). Prior to co-IP, 30 μL of total protein was collected, and 10 μL of 4 × SDS Loading Buffer

(200 mM Tris-HCl, pH 6.8; 40% glycerol; 0.04% bromophenol blue; 8% SDS; 5% β-mercaptoethanol; 4 mM DTT) was added and treated at 98 °C for 5 min to serve as input control, followed by the

addition of anti-GFP agarose beads (Chromotek Cat. gta-20) to the total protein and immunoprecipitation at 4 °C for 3 h. The agarose beads were collected and washed with washing buffer (20 

mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100) three times, and the supernatant was removed and 1 × SDS Loading Buffer was added (40 μL), then the beads were treated at

98 °C for 5 min before performing western blot assay. Immunoblotting analysis of immunoprecipitated and input proteins was performed with anti-GFP (1: 2000, GenScript) or anti-Flag

antibodies (1: 2000, Sigma-Aldrich F1804). IN VITRO PULL-DOWN ASSAY AtHIR4-His or GST-AtHIR4, MBP, and MBP-SsPEIE1 fusion proteins were expressed in _Escherichia coli_ strain BL21. The

obtained prokaryotically expressed proteins were dissolved in PBS (pH 7.4), MBP and MBP-SsPEIE1 at different concentration gradients were incubated with AtHIR4-His and GST-AtHIR4 mixed

proteins at 4 °C for 1–2 h, followed by incubation with Glutathione Resin (GenScript, Cat. No. L00206) for 1 h at 4 °C to enrich GST-AtHIR4. The beads were collected and washed three times

with PBS (5 mL). Proteins were detected with anti-His (1: 5000, abmart), anti-MBP (1: 5000, abmart) and anti-GST (1: 5000, abmart) antibodies by immunoblotting. RNA ISOLATION, CDNA

SYNTHESIS, AND RT-QPCR ANALYSIS Plant and fungal samples were ground to a powder in liquid nitrogen, total RNA was extracted using tizol, and DNA was removed using RNase-free recombinant

DNase I (Takara 2270A). To detect the expression pattern of _SsPEIE1_, the wildtype strain was cultured on PDA for 36 h, mycelium was collected and then transferred to new PDA plates or

inoculated onto leaves of rape. Leaves of 4-week-old Col-0 plants sprayed with _S. sclerotiorum_ mycelial suspension were collected at 0, 1, 3, 12, 24, and 48 h for nucleic acid extraction.

Mycelia were harvested at 0 h, 1.5 h, 3 h, 6 h, 12 h, and 24 h and 1, 2, 3, 5, and 7 days to extract nucleic acids. The concentration of total RNA was quantified using a spectrophotometer

(Thermo Fisher Scientific), and first-strand cDNA was synthesized using Easy Script One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen AE311-02). RT-PCR was performed using a CFX96

Real-Time PCR Detection System and TransStart Green qPCR SuperMix (Transgen AQ101-01). RNA samples for each real-time PCR were normalized with the _β-tubulin_ gene _Sstub1_ of _S.

sclerotiorum_ and the ubiquitin 5 gene _AtUBQ5_ for _Arabidopsis_, respectively. For each gene, real-time PCR assays were repeated at least twice, with three biological replicates each time.

Primers used are provided in Supplementary Data 2. ACCESSION NUMBERS Sequence data in this article can be found in the _S. sclerotiorum_ Database or the _Arabidopsis_ Information Resource

under the following accession numbers: _SsPEIE1_ (_Ss1G_00849_), _Sstub1_ (_Ss1G_04652_), _ATHIR1_ (_AT1G69840_), _ATHIR2_ (_AT3G01290_), _ATHIR3_ (_AT5G51570_), _ATHIR4_ (_AT5G62740_),

SL_HIR1_ (_LOC101245344_), CA_HIR1_ (_LOC107862499_), _ATUBQ5_ (_AT3G62250_), _FRK1_ (_AT2G19190_), _NHL10_ (_AT2G35980_), _WRKY30_ (_AT5G24110_), _PR1_ (_AT2G14610_), _PHI1_ (_AT2G21870_).

The _Arabidopsis_ mutant numbers are: _hir1_ (_SALK_088328C_), _hir2_ (_SALK_124393C_), _hir3_ (_SALK_104547C_), and _hir4_ (_WiscDsLox489-492B7_). STATISTICS AND REPRODUCIBILITY Data from

growth rate assay, inoculation assay, leaf area assay, and qPCR assay were expressed as mean ± standard deviation (SD), and data from ROS burst assay were expressed as mean ± standard error

of mean (SEM). Statistical analyses were performed using one-way ANOVA, and graphs were generated by GraphPad Prism 8.0 software. In this study, representative experimental results, such as

growth rate assay, inoculation assays, ROS assay, MAPKs phosphorylation assays, fluorescence observations, and any western blot analyses, were independently repeated three times, producing

similar results, with the most representative one being shown. All experimental observations were carried out without pre-selection of groups. The plants and strains used in each independent

repeated experiment were from the same batch of planting or activation, and were randomly assigned to the control group and the experimental group. No other forms of randomization were

relevant to this study. REPORTING SUMMARY Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. DATA AVAILABILITY All of our

raw data including full uncropped images in the manuscript are provided in figshare (https://doi.org/10.6084/m9.figshare.27178839). The authors declare that the other data supporting the

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Pathogen protein modularity enables elaborate mimicry of a host phosphatase. _Cell_ 186, 3196–3207 (2023). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank

Yangrong Cao of Huazhong Agricultural University for provision of pBridge vector. We also acknowledge Dengfeng Hong of Huazhong Agricultural University for providing rapeseed Xiao Yun and

genetic transformation method. This research was supported by the National Nature Science Foundation of China (32172368, 32130087), the earmarked fund of China Agriculture Research System

(CARS-12), and the Huazhong Agricultural University Scientific and Technological Self-innovation Foundation (2021ZKPY014), and US Department of Agriculture Agricultural Research Service

Sclerotinia Initiative. The funders were not involved in study design, data collection and analysis, decision to publish, or manuscript preparation. AUTHOR INFORMATION Author notes * These

authors contributed equally: Xiaofan Liu, Huihui Zhao. AUTHORS AND AFFILIATIONS * State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei Province,

China Xiaofan Liu, Huihui Zhao, Mingyun Yuan, Pengyue Li, Jiatao Xie, Bo Li, Xiao Yu, Tao Chen, Yang Lin, Daohong Jiang & Jiasen Cheng * The Provincial Key Lab of Plant Pathology of

Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei Province, China Xiaofan Liu, Huihui Zhao, Mingyun Yuan, Pengyue Li, Jiatao Xie, 

Yanping Fu, Bo Li, Xiao Yu, Tao Chen, Yang Lin, Daohong Jiang & Jiasen Cheng * United States Department of Agriculture, Agricultural Research Service, and Department of Plant Pathology,

Washington State University, Pullman, WA, USA Weidong Chen Authors * Xiaofan Liu View author publications You can also search for this author inPubMed Google Scholar * Huihui Zhao View

author publications You can also search for this author inPubMed Google Scholar * Mingyun Yuan View author publications You can also search for this author inPubMed Google Scholar * Pengyue

Li View author publications You can also search for this author inPubMed Google Scholar * Jiatao Xie View author publications You can also search for this author inPubMed Google Scholar *

Yanping Fu View author publications You can also search for this author inPubMed Google Scholar * Bo Li View author publications You can also search for this author inPubMed Google Scholar *

Xiao Yu View author publications You can also search for this author inPubMed Google Scholar * Tao Chen View author publications You can also search for this author inPubMed Google Scholar

* Yang Lin View author publications You can also search for this author inPubMed Google Scholar * Weidong Chen View author publications You can also search for this author inPubMed Google

Scholar * Daohong Jiang View author publications You can also search for this author inPubMed Google Scholar * Jiasen Cheng View author publications You can also search for this author

inPubMed Google Scholar CONTRIBUTIONS X.L. and J.C. designed experiments, analyzed the data, and wrote the manuscript; H.Z. performed gene complement, identifying, and partial vector

construction; M.Y. conducted rapeseed transformation; P.L. constructed the relevant vectors and contributed to experimental design; D.J., J.X., Y.F., B.L., X.Y., T.C. and Y.L. designed the

research and provided critical feedback; W.C. contributed to revising the manuscript. CORRESPONDING AUTHOR Correspondence to Jiasen Cheng. ETHICS DECLARATIONS COMPETING INTERESTS The authors

declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Communications_ thanks Yangdou Wei, and the other, anonymous, reviewer(s) for their contribution to the peer

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Liu, X., Zhao, H., Yuan, M. _et al._ An effector essential for virulence of necrotrophic fungi targets plant HIRs to inhibit host immunity. _Nat Commun_ 15, 9391 (2024).

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