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

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.

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 sativa3,4,5,6. 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.

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).

a Phylogenetic relationship of SsPEIE1 and its homologs from other fungi determined with the maximum-likelihood algorithm. Branch lengths are proportional to the average probability of

change for characters on that branch. The phylogeny was constructed with Mega 6.0, using the neighbor-joining method. b SsPEIE1 is a secreted protein containing 152 amino acids including a

signal peptide (SP) and four cysteine residues. c The invertase activity in TTC solution. TTC encountered sucrose breakdown products to produce triphenylformazan, which showed a red reaction

to confirm that the functional signal peptide enables secretion of the sucrose-converting enzyme. d The secretory function of the SsPEIE1 signal peptide was verified by a yeast secretion

trap screen assay. e Relative levels of SsPEIE1 transcript accumulation were determined by RT-qPCR using total RNA extracted from Arabidopsis plants inoculated with S. sclerotiorum in an

infection time course (gray columns) or from fungal culture grown on PDA plates at 20 °C (black columns). Levels of β-tubulin transcripts of S. sclerotiorum were used to normalize different

samples (n = 3 biologically repetitions). f, g Growth rates and colony morphology of wild-type (WT), ΔSsPEIE1-1, ΔSsPEIE1-2, ΔSsPEIE1-1-C1, and ΔSsPEIE1-1-C2 strains grown at 20 °C for 15

days on PDA, n = 3 biologically repetitions. h Virulence assay of S. sclerotiorum WT, ΔSsPEIE1 mutants ΔSsPEIE1-1 and ΔSsPEIE1-2, and complementary strains ΔSsPEIE1-1-C1 and ΔSsPEIE1-1-C2 on

Col-0, photographed at 40 hpi. i Lesion areas by the cross-over method in (h), n = 8 biologically independent samples. j Relative biomass in (h) analyzed by qPCR (n = 3 biologically

repetitions). Data represent means ± SD. Different letters on the same graph indicate statistical significance at p