Abstract Botrytis cinerea is a necrotrophic plant pathogen with an extremely wide host range. During invasion, the fungus induces rapid host cell death and proliferates in the necrotic tissue. The mechanisms of host killing are still incompletely understood, they involve secretion of lytic enzymes, phytotoxic metabolites and cell death inducing proteins (CDIPs). We have previously shown that the sequential knockout of up to 12 CDIPs leads to a substantial reduction of virulence of B. cinerea mutants. In this study, we have identified additional CDIPs and generated an extended mutant series culminating in a 29x mutant which is deficient in most currently known CDIPs and two phytotoxic metabolites. These mutants are strongly reduced in infection, but still induced necrosis and grey mould symptoms, demonstrating that additional determinants of host killing remain unidentified. Overexpression of the highly phytotoxic Nep1 in a 22-fold CDIP mutant failed to increase its virulence. Reevaluation of several CDIPs previously described as virulence factors revealed for most tested CDIPs no major contribution to pathogenesis. Together with the observation that none of the CDIPs are specific to B. cinerea , our data question a particular role of CDIPs for necrotrophic pathogenesis. In contrast, generation of a mutant lacking all six predicted endo-polygalacturonases confirmed their major but not exclusive role for tissue degradation and infection.

Abstract 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 necrotrophic fungus Botrytis cinerea, releases numerous phytotoxic, cell death inducing proteins (CDIPs) during infection. The precise role of these proteins and their molecular function, however, is still unknown. Here, we report on the identification of a previously unknown CDIP, the glucanosyltransferase Gas5A. Functional characterization revealed that the C-terminal 60 aa of Gas5A are sufficient to induce cell death, independent from its putative enzymatic function. Gas5A localization and functional dependence on the receptor-associated kinase suppressor of BIR1-1 (SOBIR1) and the plant defense regulator ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) indicate recognition as a pathogen-associated molecular pattern (PAMP) at the plant plasma membrane, but it is toxic also when delivered inside plant cells. Generation of a CRISPR/Cas9-assisted Botrytis knockout strain did not indicate any impact of Gas5A on virulence. Taken together, Gas5A represents a novel PAMP-like CDIP with additional intracellular phytotoxic activity.

Molecular chaperones are essential throughout a protein's life and act already during protein synthesis. Bacteria and chloroplasts of plant cells share the ribosome-associated chaperone trigger factor (Tig1 in plastids), facilitating maturation of emerging nascent polypeptides. While typical trigger factor chaperones employ three domains for their task, the here described truncated form, Tig2, contains just the ribosome binding domain. Tig2 is widely present in green plants and appears to have acquired an entirely different task than co-translational nascent polypeptide folding. Tig2 deletion results in remarkable leaf developmental defects of cold-exposed Arabidopsis thaliana plants and specific defects in plastidic ribosomes. Our data indicate that Tig2 functions during ribosome biogenesis by promoting the maturation of the large subunit. We hypothesize that Tig2 binding to the ribosomal tunnel-exit surface aids protecting this sensitive surface during assembly. Tig2 illustrates a fascinating concept of how a chaperone domain evolved individually, serving a completely different molecular task.

We are deeply saddened to report that David Gordon Robinson passed away on Tuesday, 5 November 2024. He has left behind his wife and three children. Without doubt, David was one of Europe's leading plant cell biologists and electron microscopists, best known for his research on intracellular trafficking and cellular organization. He is leaving a legacy of groundbreaking research and influence in the field. In this obituary, we want to recapitulate the most important stages from the impressive career of a truly unique character.

The phytohormone salicylic acid (SA) has a key role in regulating plant growth and stress response. In the past, most of the growth-related SA functions have been explained by crosstalk with the master growth regulator auxin. By affecting polarity of auxin transporters, SA changes auxin distribution in the root and inhibits growth of the main root and activates lateral root formation. However, only recently there is evidence emerging that SA impacts growth processes independently of nuclear auxin signalling, possessing yet unknown mechanistic functions. Here we show that SA activity depends on TRANSMEMBRANE KINASE 1 (TMK1) resulting in apoplast alkalinization and growth restriction. SA treatment prevents phosphorylation and activation of plasma membrane (PM) H+-ATPases at the cell surface and does not depend on the auxin receptor Auxin Binding Protein 1 (ABP1). We suggest that alkalinization of the apoplast by SA serves as mechanism to balance stress response and growth. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, CRC1101-A09, TRR356-B01, GR4559/4-1, SCHE 1836/4-2, SCHE 1836/5-1 CEPLAS-EXC-2048/1, 390686111 BioComp research initiative, RLP, #4

Auxin is a crucial phytohormone that regulates plant development and facilitates dynamic responses to environmental changes through subcellular control mechanisms. PIN-LIKES (PILS) are auxin transport facilitators at the endoplasmic reticulum (ER) that mediate nuclear auxin abundance and signaling. Although the posttranslational regulation of PILS is important for acclimating growth responses, the molecular mechanisms involved remain largely unknown. This study demonstrates that components of the ER-associated degradation (ERAD) machinery regulate the proteasome-dependent degradation of functional PILS proteins under nonstressed conditions. We further reveal that both internal and external signals use the ERAD complex to differentially modulate the turnover rates of PILS proteins. Our findings uncover an additional physiological role of the ERAD complex in regulating PILS protein turnover. This finding uncovers the interplay between protein homeostasis at the ER and growth regulation, opening unexplored molecular avenues into how plants acclimate to internal and external cues.

PSII assembly requires auxiliary factors, including Psb28. Although the absence of Psb28 in cyanobacteria has little effect on PSII assembly, we show here that the Chlamydomonas (Chlamydomonas reinhardtii) psb28 null mutant is severely impaired in PSII assembly, showing drastically reduced PSII supercomplexes, dimers, and monomers, while overaccumulating early PSII assembly intermediates reaction center II (RCII), CP43mod, and D1mod. The mutant had less PSI and more cytochrome b6f complex, its thylakoids were organized mainly as monolayers, and it had a distorted chloroplast morphology. Complexome profiling of the psb28 mutant revealed that THYLAKOID ENRICHED FRACTION 5 (TEF5), the homolog of Arabidopsis (Arabidopsis thaliana) PHOTOSYSTEM B PROTEIN 33/LIGHT HARVESTING-LIKE 8, comigrated particularly with RCII. TEF5 also interacted with PSI. A Chlamydomonas tef5 null mutant was severely impaired in PSII assembly and overaccumulated RCII and CP43mod. RC47 was not detectable in the light-grown tef5 mutant. Our data suggest a possible role for TEF5 in RCII photoprotection or maturation. Both the psb28 and tef5 mutants exhibited decreased synthesis of CP47 and PsbH, suggesting negative feedback regulation possibly exerted by the accumulation of RCII and/or CP43mod in both mutants. The strong effects of missing auxiliary factors on PSII assembly in Chlamydomonas suggest a more effective protein quality control system in this alga than in land plants and cyanobacteria.

While the role of salicylic acid (SA) in plant defence has been investigated for decades, its role in regulating plant growth and development has only come into focus recently. SA application inhibits growth independently of the established "Nonexpressor of Pathogenesis Related" (NPR) receptors. However, the underlying mechanism at the cellular level remains largely elusive. Here, we show that SA induces changes in vacuolar morphology and a significant increase in vacuolar pH in Arabidopsis (Arabidopsis thaliana). We demonstrate SA-mediated inhibition of V-ATPase activity, which is confirmed by experiments using the V-ATPase mutant vha-a2 vha-a3. The observed effects seem to be independent of the phytohormone auxin, which has been reported to crosstalk with SA. By inhibiting V-ATPase activity, SA impacts basic cellular functions such as vesicle trafficking and/or nutrient storage, affecting cell size and growth. Our results reveal a NPR-independent mechanism that attenuates growth, potentially reallocating resources to enhance plant robustness and promote endurance during environmental stresses.

While the role of salicylic acid (SA) for plant immunity has been investigated for decades, its function in regulating plant growth and development has only come into focus recently. Several studies indicate that SA – auxin crosstalk plays an important role in mediating SA-induced effects. However, not all findings can be explained by this crosstalk alone and SA-specific effects on intracellular organization have been reported such as inhibition of endocytosis and changes of vacuolar pH and morphology. Notably, several SA-related functions seem to be independent of the SA receptors Nonexpressor of Pathogenesis-Related genes (NPRs). This review summarizes the effects of SA on intracellular organization and predicts the existence of as yet unknown signaling pathways to explain the current findings. We provide a short general introduction including SA biosynthesis and SA signaling and address how NPR-independent intracellular changes necessitate specific signaling to regulate growth and development.

While Botrytis cinerea causes gray mold on many plants, its close relative, Botrytis fabae, is host-specifically infecting predominantly faba bean plants. To explore the basis for its narrow host range, a gapless genome sequence of B. fabae strain G12 (BfabG12) was generated. The BfabG12 genome encompasses 45.0 Mb, with 16 chromosomal telomere-to-telomere contigs that show high synteny and sequence similarity to the corresponding B. cinerea B05.10 (BcB0510) chromosomes. Compared to BcB0510, it is 6% larger, due to many AT-rich regions containing remnants of transposable elements, but encodes fewer genes (11,420 vs. 11,707), due to losses of chromosomal segments with up to 20 genes. The coding capacity of BfabG12 is further reduced by nearly 400 genes that had been inactivated by mutations leading to truncations compared to their BcB0510 orthologues. Several species-specific gene clusters for secondary metabolite biosynthesis with stage-specific expression were identified. Comparison of the proteins secreted during infection revealed high similarities, including 17 phytotoxic proteins that were detected in both species. Our data indicate that evolution of the host-specific B. fabae occurred from an ancestral pathogen with wide host range similar to B. cinerea and was accompanied by losses and degeneration of genes, thereby reducing its pathogenic flexibility.

Several auxiliary factors are required for the assembly of photosystem (PS) II, one of which is Psb28. While the absence of Psb28 in cyanobacteria has little effect on PSII assembly, we show here that the Chlamydomonas psb28 -null mutant is severely impaired in PSII assembly, showing drastically reduced PSII supercomplexes, dimers and monomers, while overaccumulating RCII, CP43mod and D1mod. The mutant had less PSI and more Cyt b6f and showed fewer thylakoid stacks and distorted chloroplast morphology. Complexome profiling of the psb28 mutant revealed that TEF5, the homolog of Arabidopsis PSB33/LIL8, co-migrated particularly with RCII. TEF5 also interacted with PSI. A Chlamydomonas tef5 null mutant is also severely impaired in PSII assembly and overaccumulates RCII and CP43mod. RC47 was not detectable in the light-grown tef5 mutant. Our data suggest a possible role for TEF5 in facilitating the assembly of CP47mod into RCII. Both the psb28 and tef5 mutants exhibited decreased synthesis of CP47 and PsbH, suggesting negative feedback regulation possibly exerted by the accumulation of RCII and/or CP43mod in both mutants. The strong effects of missing auxiliary factors on PSII assembly in Chlamydomonas suggest a more effective protein quality control system in this alga than in land plants and cyanobacteria. One-sentence summary The Chlamydomonas psb28 mutant is severely impaired in PSII assembly which via complexome profiling allowed identifying TEF5 as a novel PSII assembly factor that likely facilitates CP47 assembly. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors () is: Michael Schroda (m.schroda{at}rptu.de).

Filamentous pathogens need to overcome plant barriers for successful infection. To this end, special structures, most commonly appressoria, are used for penetration. In differentiated appressoria, the generation of high turgor pressure is mandatory to breach plant cell wall and cuticle. However, quantitative description of turgor pressure and resulting invasive forces are only described for a handful of plant pathogens. Recent advances in methodology allowed determination of surprisingly high pressures and corresponding forces in oomycetes and a necrotrophic fungus. Here, we describe turgor generation in appressoria as essential function for host penetration. We summarize the known experimentally determined turgor pressure as well as invasive forces and discuss their universal role in plant pathogen infection.

Summary The devastating pathogen Botrytis cinerea infects a broad spectrum of host plants, causing great socio‐economic losses. The necrotrophic fungus rapidly kills plant cells, nourishing their wall and cellular contents. To this end, necrotrophs secrete a cocktail of cell wall degrading enzymes, phytotoxic proteins and metabolites. Additionally, many fungi produce specialized invasion organs that generate high invasive pressures to force their way into the plant cell. However, for most necrotrophs, including Botrytis, the biomechanics of penetration and its contribution to virulence are poorly understood. Here, we use a combination of quantitative micromechanical imaging and CRISPR–Cas‐guided mutagenesis to show that Botrytis uses substantial invasive pressure, in combination with strong surface adherence, for penetration. We found that the fungus establishes a unique mechanical geometry of penetration that develops over time during penetration events, and which is actin cytoskeleton dependent. Furthermore, interference of force generation by blocking actin polymerization was found to decrease Botrytis virulence, indicating that also for necrotrophs, mechanical pressure is important in host colonization. Our results demonstrate for the first time mechanistically how a necrotrophic fungus such as Botrytis employs this ‘brute force’ approach, in addition to the secretion of lytic proteins and phytotoxic metabolites, to overcome plant host resistance.

The phytohormone salicylic acid (SA) is a key factor to balance plant defence as well as growth and development. While its role in plant defence has been investigated for decades, regulation of plant growth and development has only come into focus recently. It has been demonstrated that SA application inhibits growth independently of the established Non-expressor of Pathogenesis Related (NPR) receptors. However, the underlying mechanism of this growth inhibition on the cellular level remains largely elusive. Here we show that SA restricts cell elongation and induces changes of vacuolar morphology and pH. Rapidly upon SA application we observe homotypic vacuole fusion and a significant increase in vacuolar pH. These changes seem to be independent of the phytohormone auxin which has been reported to crosstalk with SA. By increasing vacuolar pH, SA directly impacts basic cellular functions such as vesicle trafficking or nutrient storage, leading eventually to cell size restriction and limited growth. Our results demonstrate an NPR-independent mechanism to attenuate growth, potentially allowing free resources to be relocated to withstand environmental stresses. ![Figure][1] Graphical abstract Salicylic Acid (SA) triggers a reduction in cell size and leads to a spherical vacuolar phenotype. This morphological change is accompanied by an increase in vacuolar pH, potentially due to the enhanced activity of a glycosylated SA (SAG) H+-antiporter and/or the inhibition of V-ATPase activity. In addition, SA disrupts the polarity of PIN2 auxin transporters, resulting in their uniform distribution across the cell membrane. ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

VPS13 are conserved lipid transporters with multiple subcellular localizations playing key roles in many fundamental cellular processes. While the localization and function of VPS13 have been extensively investigated in yeast and animals, little is known about their counterparts in plants, particularly regarding their role in stress response. In this study, we characterized AtVPS13M1, one of the four VPS13 paralogs of the flowering plant Arabidopsis thaliana . We show that AtVPS13M1 binds and transports glycerolipids with a low specificity in vitro . AtVPS13M1 interferes with phospholipids degradation in response to phosphate starvation, a nutrient stress that triggers a massive remodeling of membrane lipids. AtVPS13M1 is mainly expressed in young dividing and vascular tissues. Finally, we show that AtVPS13M1 is mainly located at the surface of mitochondria in leaves. Overall, our work highlights the conserved role in lipid transport of VPS13 in plants, reveals their importance in nutrient stress response and opens important perspectives for the understanding of lipid remodeling mechanisms and for the characterization of this protein family in plants.### Competing Interest StatementThe authors have declared no competing interest.

Plant vacuoles play key roles in cellular homeostasis performing catabolic and storage functions, regulating pH and ion balance. The essential role of vacuoles for plant cell viability makes them a notoriously difficult subject to study impeding reaching the consensus on the mechanism of vacuolar establishment and the source of membrane material for it. Our previous suggestion of endoplasmic reticulum being the main membrane contributor for the tubular network of young vacuoles was recently challenged in a study proposing that young plant vacuoles comprise a set of individual vesicles that are formed de novo via homotypic fusion of multivesicular bodies (MVBs). To resolve these seemingly contradictory observations we have carefully revaluated both hypotheses. Here we provide a systematic overview of successive vacuolar biogenesis stages in Arabidopsis root, starting from the youngest cells proximate to the quiescent center. We validate our previous conclusions by demonstrating that the vacuolar dye BCECF is fully suitable for studying the organelle’s morphology and provide 3D models of vacuoles at all developmental stages. Furthermore, we established a customized FRAP assay and proved that even at the earliest stages of biogenesis, vacuoles comprise a connected network. Finally, we summarized the new and pre-existing evidence substantiating that vacuolar structures cannot originate solely from MVBs.

According to their lifestyle, plant pathogens are divided into biotrophic and necrotrophic organisms. Biotrophic pathogens exclusively nourish living host cells, whereas necrotrophic pathogens rapidly kill host cells and nourish cell walls and cell contents. To this end, the necrotrophic fungus Botrytis cinerea secretes large amounts of phytotoxic proteins and cell wall-degrading enzymes. However, the precise role of these proteins during infection is unknown. Here, we report on the identification and characterization of the previously unknown toxic protein hypersensitive response-inducing protein 1 (Hip1), which induces plant cell death. We found the adoption of a structurally conserved folded Alternaria alternata Alt a 1 protein structure to be a prerequisite for Hip1 to exert its necrosis-inducing activity in a host-specific manner. Localization and the induction of typical plant defense responses by Hip1 indicate recognition as a pathogen-associated molecular pattern at the plant plasma membrane. In contrast to other secreted toxic Botrytis proteins, the activity of Hip1 does not depend on the presence of the receptor-associated kinases BRI1-associated kinase 1 and suppressor of BIR1-1. Our results demonstrate that recognition of Hip1, even in the absence of obvious enzymatic or pore-forming activity, induces strong plant defense reactions eventually leading to plant cell death. Botrytis hip1 overexpression strains generated by CRISPR/Cas9 displayed enhanced infection, indicating the virulence-promoting potential of Hip1. Taken together, Hip1 induces a noncanonical defense response which might be a common feature of structurally conserved fungal proteins from the Alt a 1 family.

In the cytosol of plant cells, heat-induced protein aggregates are resolved by the CASEIN LYTIC PROTEINASE/HEAT SHOCK PROTEIN 100 (CLP/HSP100) chaperone family member HSP101, which is essential for thermotolerance. For the chloroplast family member CLPB3 this is less clear, with controversial reports on its role in conferring thermotolerance. To shed light on this issue, we have characterized two clpb3 mutants in Chlamydomonas reinhardtii. We show that chloroplast CLPB3 is required for resolving heat-induced protein aggregates containing stromal TRIGGER FACTOR (TIG1) and the small heat shock proteins 22E/F (HSP22E/F) in vivo, and for conferring thermotolerance under heat stress. Although CLPB3 accumulation is similar to that of stromal HSP70B under ambient conditions, we observed no prominent constitutive phenotypes. However, we found decreased accumulation of the PLASTID RIBOSOMAL PROTEIN L1 (PRPL1) and increased accumulation of the stromal protease DEG1C in the clpb3 mutants, suggesting that a reduction in chloroplast protein synthesis capacity and an increase in proteolytic capacity may compensate for loss of CLPB3 function. Under ambient conditions, CLPB3 was distributed throughout the chloroplast, but reorganized into stromal foci upon heat stress, which mostly disappeared during recovery. CLPB3 foci were localized next to HSP22E/F, which accumulated largely near the thylakoid membranes. This suggests a possible role for CLPB3 in disentangling protein aggregates from the thylakoid membrane system.