Correction: Bleyer et al. Together for the Better: Improvement of a Model Based Strategy for Grapevine Downy Mildew Control by Addition of Potassium Phosphonates. Plants2020, 9, 710.

Error in Figure [...].

A method for phenotypic evaluation of grapevine resistance in relation to phenological development.

Fungus-resistant grapevine cultivars, so called PIWIs, are characterized by increased resistance to powdery mildew and downy mildew. However, in order to maintain the durability of resistance in these new grape cultivars, targeted fungicide treatments are recommended. For ideal schedule of these treatments, it is necessary to recognize the most sensitive organs of the grape. This study introduces a method for phenotypic evaluation of Plasmopara viticola resistance in grape clusters under controlled and standardized conditions during phenological development over the entire season. The approach was validated with the traditional cultivar Pinot Noir and the PIWIs Cabernet Cortis (Rpv3.3, Rpv10), Solaris (Rpv3.3, Rpv10) and Souvignier Gris (Rpv3.2). All cultivars were susceptible during the early stages of development up to flowering, and resistance levels increased as phenological development progressed. Cabernet Cortis and Solaris clusters were susceptible until fruit development (BBCH 71-73) when they became almost completely resistant. No differences between Souvignier Gris and Pinot Noir were detected until berries were pea-sized (BBCH 75) when P. viticola resistance of Souvignier Gris clusters increased significantly. Ontogenetic resistance in Pinot Noir was detected at berry touch (BBCH 77-79) and clusters of this cultivar were almost completely resistant at the beginning of ripening (BBCH 81-83). These results indicate that the approach presented is suitable for determining the resistance of grape cultivars at different stages of development. Consequently, in the future, fungicide applications can be adjusted more precisely to the resistance level of a grape cultivar during the growing season.

Sequence and Gene Expression Analysis of Recently Identified NLP from Plasmopara viticola.

Grapevine downy mildew, evoked by the obligate biotrophic oomycete Plasmopara viticola, is one of the most challenging diseases in viticulture. P. viticola establishes an infection by circumvention of plant immunity, which is achieved by the secretion of effector molecules. One family of potential effectors are the necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP). NLP are most abundant in plant pathogenic microorganisms and exist in cytotoxic and non-cyctotoxic forms. Cytotoxic NLP often act as virulence factors and are synthesized in necrotrophic or hemibiotrophic pathogens during the transition from biotrophic to necrotrophic growth. In addition to these cytotoxic NLP, many non-cytotoxic NLP have been identified; their function in biotrophic pathogens is still unknown. In 2020, eight different NLP coding genes were identified in P. viticola and named PvNLP1 to PvNLP8 (Plasmopara viticolaNLP 1-8). In the present study, PvNLP4 to PvNLP8 were characterized by using qPCR analysis and transient expression in the model plant Nicotiana benthamiana. Gene expression analysis showed high PvNLP expression during the early stages of infection. Necrosis-inducing activity of PvNLP was not observed in the nonhost N. benthamiana.

Studies on the Occurrence of Viruses in Planting Material of Grapevines in Southwestern Germany.

Viral diseases in viticulture lead to annual losses in the quantity and quality of grape production. Since no direct control measures are available in practice, preventive measures are taken to keep the vines healthy. These include, for example, the testing of propagation material for viruses such as Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV) or Grapevine leafroll-associated virus 1 (GLRaV-1) and 3 (GLRaV-3). As long-term investigations have shown, GLRaV-1 (2.1%) occurs most frequently in southwestern German wine-growing regions, whereas GLRaV-3 (<0.1%) is almost never found. However, tests conducted over 12 years indicate that there is no general decline in virus-infected planting material. Thus, it can be assumed that a spread of the viruses via corresponding vectors still takes place unhindered. Beyond the examinations regulated within the German Wine Growing Ordinance, one-time tests were carried out on Grapevine Pinot gris virus (GPGV). This analysis showed that GPGV was found in 17.2% of the samples.

Together for the Better: Improvement of a Model Based Strategy for Grapevine Downy Mildew Control by Addition of Potassium Phosphonates.

Grapevine downy mildew is one of the major diseases in viticulture. To control this disease, a more effective strategy has been developed and established based on growth and model data as well as on a combination of fungicides. For this purpose, the systemic plant protection product potassium phosphonate (PP) was combined with two contact fungicides. Treatments were carried out according to the different experimental conditions after the growth of 400 cm2, 600 cm2, and 800 cm2 leaf area per primary shoot. PP increased the effectiveness of the preventive fungicides whenever high infection pressure was the case. The experiments also show that it is possible to extend the treatment intervals from 400 cm2 to 600 cm2 new leaf area when PP was added. However, none of the tested treatments were sufficient for the extension to intervals of 800 cm2. These data show that PP can be a key factor in the reduction of the application of synthetic or copper-based fungicides.

Identification and Characterization of Nep1-Like Proteins From the Grapevine Downy Mildew Pathogen Plasmopara viticola.

The obligate biotrophic oomycete Plasmopara viticola causes tremendous problems in viticulture by evoking grapevine downy mildew. P. viticola, like other plant pathogens, achieves infection by suppression of plant innate immunity by secretion of effector molecules into its host plant. An ever-expanding family of proteins with effector-like characteristics is formed by the "Necrosis and Ethylene inducing peptide 1 (Nep1)-like proteins" (NLPs). NLPs can be divided into two groups by their ability to induce necrosis. While cytotoxic NLPs may act as virulence factors for a necrotrophic or hemibiotrophic plant pathogen, the role of non-cytotoxic NLPs is so far unknown. In this study, we identified eight independent NLPs in P. viticola and selected three for functional analysis. While one was identified as a putative pseudo gene, two contain all so far described critical key elements for necrosis formation except for an N-terminal signal peptide. Further characterization revealed that none of the putative necrosis elicitors was able to actually induce necrosis, neither in several susceptible or resistant Vitis species nor in the dicot model plant Nicotiana benthamiana. This inability exists independently of the presence or absence of a signal peptide. However, any possible mechanism for the suppression of the ability to induce necrosis in planta was not detected. Interestingly, expression analysis of the presumed pseudo gene revealed remarkable differences between pure sporangia solution and sporangia in the presence of leaf material. To our knowledge, this is the first report of this kind of regulation that suggests an important function of so far nonfunctional "pseudo" NLP genes during the first hours of infection.

Moonlighting Function of Phytochelatin Synthase1 in Extracellular Defense against Fungal Pathogens.

Phytochelatin synthase (PCS) is a key component of heavy metal detoxification in plants. PCS catalyzes both the synthesis of the peptide phytochelatin from glutathione and the degradation of glutathione conjugates via peptidase activity. Here, we describe a role for PCS in disease resistance against plant pathogenic fungi. The pen4 mutant, which is allelic to cadmium insensitive1 (cad1/pcs1) mutants, was recovered from a screen for Arabidopsis mutants with reduced resistance to the nonadapted barley fungal pathogen Blumeria graminis f. sp. hordei PCS1, which is found in the cytoplasm of cells of healthy plants, translocates upon pathogen attack and colocalizes with the PEN2 myrosinase on the surface of immobilized mitochondria. pcs1 and pen2 mutant plants exhibit similar metabolic defects in the accumulation of pathogen-inducible indole glucosinolate-derived compounds, suggesting that PEN2 and PCS1 act in the same metabolic pathway. The function of PCS1 in this pathway is independent of phytochelatin synthesis and deglycination of glutathione conjugates, as catalytic-site mutants of PCS1 are still functional in indole glucosinolate metabolism. In uncovering a peptidase-independent function for PCS1, we reveal this enzyme to be a moonlighting protein important for plant responses to both biotic and abiotic stresses.

The barley (Hordeum vulgare) cellulose synthase-like D2 gene (HvCslD2) mediates penetration resistance to host-adapted and nonhost isolates of the powdery mildew fungus.

Cell walls and cellular turgor pressure shape and suspend the bodies of all vascular plants. In response to attack by fungal and oomycete pathogens, which usually breach their host's cell walls by mechanical force or by secreting lytic enzymes, plants often form local cell wall appositions (papillae) as an important first line of defence. The involvement of cell wall biosynthetic enzymes in the formation of these papillae is still poorly understood, especially in cereal crops. To investigate the role in plant defence of a candidate gene from barley (Hordeum vulgare) encoding cellulose synthase-like D2 (HvCslD2), we generated transgenic barley plants in which HvCslD2 was silenced through RNA interference (RNAi). The transgenic plants showed no growth defects but their papillae were more successfully penetrated by host-adapted, virulent as well as avirulent nonhost isolates of the powdery mildew fungus Blumeria graminis. Papilla penetration was associated with lower contents of cellulose in epidermal cell walls and increased digestion by fungal cell wall degrading enzymes. The results suggest that HvCslD2-mediated cell wall changes in the epidermal layer represent an important defence reaction both for nonhost and for quantitative host resistance against nonadapted wheat and host-adapted barley powdery mildew pathogens, respectively.

Immobilized Subpopulations of Leaf Epidermal Mitochondria Mediate PENETRATION2-Dependent Pathogen Entry Control in Arabidopsis.

The atypical myrosinase PENETRATION2 (PEN2) is required for broad-spectrum invasion resistance to filamentous plant pathogens. Previous localization studies suggested PEN2-GFP association with peroxisomes. Here, we show that PEN2 is a tail-anchored protein with dual-membrane targeting to peroxisomes and mitochondria and that PEN2 has the capacity to form homo-oligomer complexes. We demonstrate pathogen-induced recruitment and immobilization of mitochondrial subpopulations at sites of attempted fungal invasion and show that mitochondrial arrest is accompanied by peripheral accumulation of GFP-tagged PEN2. PEN2 substrate production by the cytochrome P450 monooxygenase CYP81F2 is localized to the surface of the endoplasmic reticulum, which focally reorganizes close to the immobilized mitochondria. Exclusive targeting of PEN2 to the outer membrane of mitochondria complements the pen2 mutant phenotype, corroborating the functional importance of the mitochondrial PEN2 protein subpool for controlled local production of PEN2 hydrolysis products at subcellular plant-microbe interaction domains. Moreover, live-cell imaging shows that mitochondria arrested at these domains exhibit a pathogen-induced redox imbalance, which may lead to the production of intracellular signals.

Impaired sterol ester synthesis alters the response of Arabidopsis thaliana to Phytophthora infestans.

Non-host resistance of Arabidopsis thaliana against Phytophthora infestans, the causal agent of late blight disease of potato, depends on efficient extracellular pre- and post-invasive resistance responses. Pre-invasive resistance against P. infestans requires the myrosinase PEN2. To identify additional genes involved in non-host resistance to P. infestans, a genetic screen was performed by re-mutagenesis of pen2 plants. Fourteen independent mutants were isolated that displayed an enhanced response to Phytophthora (erp) phenotype. Upon inoculation with P. infestans, two mutants, pen2-1 erp1-3 and pen2-1 erp1-4, showed an enhanced rate of mesophyll cell death and produced excessive callose deposits in the mesophyll cell layer. ERP1 encodes a phospholipid:sterol acyltransferase (PSAT1) that catalyzes the formation of sterol esters. Consistent with this, the tested T-DNA insertion lines of PSAT1 are phenocopies of erp1 plants. Sterol ester levels are highly reduced in all erp1/psat1 mutants, whereas sterol glycoside levels are increased twofold. Excessive callose deposition occurred independently of PMR4/GSL5 activity, a known pathogen-inducible callose synthase. A similar formation of aberrant callose deposits was triggered by the inoculation of erp1 psat1 plants with powdery mildew. These results suggest a role for sterol conjugates in cell non-autonomous defense responses against invasive filamentous pathogens.

Mechanisms of functional specificity among plasma-membrane syntaxins in Arabidopsis.

Syntaxins and interacting SNARE proteins enable membrane fusion in diverse trafficking pathways. The Arabidopsis SYP1 family of plasma membrane-localized syntaxins comprises nine members, of which KNOLLE and PEN1 play specific roles in cytokinesis and innate immunity, respectively. To identify mechanisms conferring specificity of action, we examined one member of each subfamily-KNOLLE/SYP111, PEN1/SYP121 and SYP132-in regard to subcellular localization, dynamic behavior and complementation of knolle and pen1 mutants when expressed from the same promoters. Our results suggest that cytokinesis-specific syntaxin requires high-level accumulation during cell-plate formation, which necessitates de novo synthesis rather than endocytosis of pre-made protein from the plasma membrane. In contrast, syntaxin in innate immunity does not need upregulation of expression but instead requires pathogen-induced and endocytosis-dependent retargeting to the infection site. This feature of PEN1 is not afforded by SYP132. Additionally, PEN1 could not substitute for KNOLLE because of SNARE domain differences, as revealed by protein chimeras. In contrast, SYP132 was able to rescue knolle as did KNOLLE-SYP132 chimeras. Unlike KNOLLE and PEN1, which appear to have evolved to perform specialized functions, SYP132 stably localized at the plasma membrane and thus might play a role in constitutive membrane fusion.

Live and let die--Arabidopsis nonhost resistance to powdery mildews.

The term "nonhost resistance" (NHR) describes the phenomenon that an entire plant species is resistant to all genetic variants of a non-adapted pathogen species. In nature, NHR represents the most robust form of plant immunity and is therefore of scientific as well as economic importance. Due to its highly complex nature, NHR has previously not been studied in detail. Recently, the establishment of model interaction systems utilizing Arabidopsis and non-adapted powdery mildews allowed the identification of several key components and conceptual conclusions. It is now generally accepted that NHR of Arabidopsis to powdery mildews comprises two distinct layers of defence: pre-invasion entry control at the cell periphery and post-invasion resistance based on cell death execution. The timely production and localised discharge of toxic compounds at sites of fungal attack appear to be pivotal for entry control. This process requires proteins involved in secretion and trans-membrane transport, synthesis and activation of indolic glucosinolates as well as gene regulation and post-translational protein modification. Post-invasion defence relies on lipase-like proteins and salicylic acid signalling. To what extent pathogen-associated molecular pattern- or effector-triggered immunity contribute to NHR remains to be investigated and is likely to depend on the model system studied.

Protein mislocalization in plant cells using a GFP-binding chromobody.

A key challenge in cell biology is to directly link protein localization to function. The green fluorescent protein (GFP)-binding protein, GBP, is a 13-kDa soluble protein derived from a llama heavy chain antibody that binds with high affinity to GFP as well as to some GFP variants such as yellow fluorescent protein (YFP). A GBP fusion to the red fluorescent protein (RFP), a molecule termed a chromobody, was previously used to trace in vivo the localization of various animal antigens. In this study, we extend the use of chromobody technology to plant cells and develop several applications for the in vivo study of GFP-tagged plant proteins. We took advantage of Agrobacterium tumefaciens-mediated transient expression assays (agroinfiltration) and virus expression vectors (agroinfection) to express functional GBP:RFP fusion (chromobody) in the model plant Nicotiana benthamiana. We showed that the chromobody is effective in binding GFP- and YFP-tagged proteins in planta. Most interestingly, GBP:RFP can be applied to interfere with the function of GFP fusion protein and to mislocalize (trap) GFP fusions to the plant cytoplasm in order to alter the phenotype mediated by the targeted proteins. Chromobody technology, therefore, represents a new alternative technique for protein interference that can directly link localization of plant proteins to in vivo function.

Arabidopsis non-host resistance to powdery mildews.

Immunity of an entire plant species against all genetic variants of a particular parasite is referred to as non-host resistance. Although non-host resistance represents the most common and durable form of plant resistance in nature, it has thus far been poorly understood at the molecular level. Recently, novel model systems have established the first mechanistic insights. The genetic dissection of Arabidopsis non-host resistance to non-adapted biotrophic powdery mildew fungi provided evidence for functionally redundant but operationally distinct pre- and post-invasion immune responses. Conceptually, these complex and successive defence mechanisms explain the durable and robust nature of non-host resistance. Pathogen lifestyle and infection biology, ecological parameters and the evolutionary relationship of the interaction partners determine differences and commonalities in other model systems.