An extended N-terminus restrains the plant cell death-inducing ability of the catalytically competent ribonuclease domain in a pea powdery mildew RALPH effector

Two homologous Alt a1-like fungal proteins possess dual activities in HIR-associated immune signaling and EDS1-dependent cell death

Necrotrophic fungi secrete numerous Cell Death-Inducing Proteins (CDIPs) that manipulate host immunity to promote disease, yet the signaling pathways underlying their phytotoxic activity remain poorly understood. Here, we identify the Botrytis cinerea Hypersensitive response-inducing protein 1 (Hip1) as a close homolog of the recently described Sclerotinia sclerotiorum effector Plant Early Immunosuppressive Effector 1 (PEIE1) and investigate the molecular basis of its activity. HIP1 and PEIE1 share high sequence similarity and a conserved AlphaFold-predicted Alt a1-like fold, they interact with the Arabidopsis plasma membrane protein HIR4, and they induce strong necrosis in Nicotiana benthamiana. Despite their high structural similarity, Hip1 and PEIE1 differ in their reported roles during fungal infection. Unexpectedly, Hip1-induced cell death requires the central immune regulator ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) as well as the downstream helper NLR network comprising ADR1 and NRG1. Together, our findings establish Hip1 as a closely related homolog of PEIE1 and suggest that these closely related Alt a1-like proteins possess dual activities: modulation of HIR-associated immune signaling and activation of EDS1-dependent host cell death.

The Conserved N-Terminal Extension of AtKEA1 Is Largely Dispensable for Plastid Function but Contributes to Potassium Homeostasis

SDJ, a pollen-expressed type III J-protein in Prunus, directly binds S-RNase and promotes recruitment of SLFL6 to an S-RNase-associated complex

In Prunus, self-incompatibility (SI) is controlled by S-RNases and pollen-expressed F-box proteins, whereas the molecular processes governing S-RNase regulation in pollen remain incompletely understood. Here, we characterized PavSDJ, a novel pollen protein from sweet cherry (Prunus avium), as a candidate factor involved in pollen-side S-RNase-associated processes. Sequence and structural analyses identified PavSDJ as a type III J-protein. Phylogenetic analyses placed PavSDJ within a distinct SDJ-like sublineage of the type III J-protein group, separate from a closely related sister lineage. Consistent with this divergence, PavSDJ was strongly expressed in anthers and pollen, whereas its sister gene was broadly expressed across organs. Transient expression assays showed that PavSDJ–GFP exhibited a predominantly cell-peripheral fluorescence pattern consistent with intracellular localization. Biochemical analyses showed that PavSDJ associated with recombinant PavS-RNases in pollen extracts and in reconstituted pull-down assays, without obvious allele preference. Proteomic analysis of PavSDJ co-immunoprecipitants from pollen extracts identified a complex including PavSLFL6 and PavSSK1. Reconstitution assays further showed that PavSDJ promoted the co-precipitation of PavSLFL6 with S-RNase. These findings identify PavSDJ as a candidate pollen-side factor in the Prunus SI pathway and provide evidence that a specialized J-protein may contribute to SI-related protein complex assembly. PavSDJ is a pollen-expressed type III J-protein that binds S-RNase and promotes recruitment of SLFL6 to an S-RNase-associated complex in Prunus. It may function as a general modifier involved in the GSI system of Prunus.

Structural modelling uncovers diverse predicted transcriptional and post-transcriptional modulators among type III effectors of symbiotic Rhizobia

Rhizobia are soil bacteria that establish nitrogen-fixing symbioses with legumes. While many rhizobia use a Type III Secretion System to deliver “Nodulation Outer Protein” (Nop) effectors, some uniquely use these proteins to initiate nodule organogenesis, bypassing classical signalling. The molecular functions of these effectors remain largely unknown due to extreme sequence divergence. Using AlphaFold2-mediated structural proteomics, we identified a modular architecture in rhizobial effectors composed of 22 distinct structural units. We reveal that many Nop effectors are cryptic transcriptional or post-transcriptional regulators, harbouring unrecognised nucleic acid–binding modules and RNA-dependent RNA polymerase domains. Crucially, these modules are conserved in specific plant pathogens, such as gall-inducing Pantoea, where our predicted structural units align with experimentally validated DNA-binding domains. Furthermore, we discovered the BPN (B3 and PUA-like nucleic acid binding) domain as a structural mimic of plant B3-domain transcription factors, pointing to a direct mechanism for hijacking legume development. Our findings strongly suggest that rhizobia employ a modular domain-fusion strategy to act as direct genetic modulators, uncovering a conserved mechanism used by both symbionts and pathogens to hijack host developmental programmes.

Functional and transcriptomic analyses in Neurospora crassa reveal a crucial role for the N-glycoprotein deglycosylation process in fungal homeostasis.

N-glycosylation is an essential post-translational modification required for proper protein folding, stability, trafficking, and secretion in eukaryotes. In such organisms, efficient endoplasmic reticulum (ER) quality control, such as that provided by the ER-associated degradation (ERAD) pathway, is critical for maintaining cellular homeostasis. During ERAD, terminally misfolded glycoproteins undergo N-deglycosylation prior to proteasomal degradation, a process typically mediated by peptide N-glycanase (PNGase). However, in filamentous fungi, the PNGase seems to be catalytically inactive, indicating evolutionary divergence from the canonical PNGase pathway. Filamentous fungi also encode endo-β-n-acetylglucosaminidases (ENGases), particularly members of glycoside hydrolase family 18 (GH18), which may compensate for the loss of canonical PNGase activity. Here, we investigated the roles of the cytosolic GH18 ENGase and a putative acidic PNGase in N. crassa using transcriptomic and functional approaches. Our results demonstrate that the cytosolic GH18 ENGase is an active deglycosylating enzyme likely associated with the ERAD pathway, whereas no deglycosylation activity was detected for the acidic PNGase. Deletion of the cytosolic ENGase severely compromises tolerance to diverse stress conditions and induces substantial transcriptomic reprogramming, including upregulation of a GH20 exo-β-n-acetylhexosaminidase under ER stress. These findings identify the cytosolic ENGase as a key component of fungal proteostasis and suggest that N. crassa activates alternative compensatory mechanisms to maintain protein quality control when canonical deglycosylation pathways are impaired.

Genome-guided discovery of clavaic acid, a decalin-containing polyketide encoded by a LovB-like polyketide synthase

Abstract Fungal iterative type I polyketide synthases (iPKSs) generate structurally diverse natural products. A subset of these assembly line enzymes, exemplified by LovB from Aspergillus terreus, contains a C-terminal condensation domain bearing a noncanonical HRxxxDG motif. While these LovB-like iPKSs are widely distributed in fungi, the majority remain uncharacterized, leaving both their associated polyketide products and the function of their unusual condensation domains largely unexplored. Here, we report the characterization of a LovB-like iPKS from Aspergillus clavatus. Heterologous reconstitution of this PKS system in Aspergillus nidulans led to the discovery of clavaic acid, a previously undescribed polyketide featuring a trimethylated trans-decalin core and an all-E-configured carboxytriene side chain. Functional analysis of the condensation domain in vivo demonstrated that it is essential for clavaic acid biosynthesis. Surprisingly, whereas the conserved arginine residue within the HRxxxDG motif was dispensable for product formation, the conserved aspartate residue was strictly required. These findings expand our understanding of LovB-like iPKSs and establish this enzyme family as a promising source of cryptic fungal polyketides. One-sentence summary Genome-guided discovery of a trans-decalin-containing polyketide encoded by a LovB-like polyketide synthase in Aspergillus clavatus.