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Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component.

Plasma membrane-resident receptor kinases (RKs) initiate signaling pathways important for plant immunity and development. In Arabidopsis (Arabidopsis thaliana), the receptor for the elicitor-active peptide epitope of bacterial flagellin, flg22, is encoded by FLAGELLIN SENSING2 (FLS2), which promotes plant immunity. Despite its relevance, the molecular components regulating FLS2-mediated signaling remain largely unknown. We show that plasma membrane ARABIDOPSIS-ALJTOINHIBITED Ca²⁺-ATPase (ACA8) forms a complex with FLS2 in planta. ACA8 and its closest homolog ACA10 are required for limiting the growth of virulent bacteria. One of the earliest flg22 responses is the transient increase of cytosolic Ca²⁺ ions, which is crucial for many of the well-described downstream responses (e.g. generation of reactive oxygen species and the transcriptional activation of defense-associated genes). Mutant aca8 aca10 plants show decreased flg22-induced Ca²⁺ and reactive oxygen species bursts and exhibit altered transcriptional reprogramming. In particular, mitogen-activated protein kinase-dependent flg22-induced gene expression is elevated, whereas calcium-dependent protein kinase-dependent flg22-induced gene expression is reduced. These results demonstrate that the fine regulation of Ca²⁺ fluxes across the plasma membrane is critical for the coordination of the downstream microbe-associated molecular pattern responses and suggest a mechanistic link between the FLS2 receptor complex and signaling kinases via the secondary messenger Ca²⁺. ACA8 also interacts with other RKs such as BRI1 and CLV1 known to regulate plant development, and both aca8 and aca10 mutants show morphological phenotypes, suggesting additional roles for ACA8 and ACA10 in developmental processes. Thus, Ca²⁺ ATPases appear to represent general regulatory components of RK-mediated signaling pathways.

Tomato production is an important part of the Swiss vegetable production with most tomato crops grown in greenhouses. Tomato plants are vulnerable to diseases caused by viruses, which can have significant impacts on crop production. This study reports the first detection of tomato fruit botch virus (ToFBV, Blunervirus solani) in Switzerland, from a tomato production site at the southern part of the Ticino region. The symptoms observed indicated presence of a viral pathogen, but tests against the most common tomato viruses were negative. Immunocapture of double-stranded RNA and its subsequent sequencing on a Flongle flowcell (Oxford Nanopore Technologies) identified the presence of ToFBV and southern tomato virus. The genome of the Swiss ToFBV isolate was very similar to that available in GenBank. Datamining of the sequence read archives found the virus in two other countries, with a highly conserved genome. With this study, there are now 12 near-complete genomes of ToFBV available, and the virus is recorded from ten countries. This study underlines the importance of continuous monitoring and research on emerging viruses in tomato production.

Emerald ash borer (EAB; Agrilus planipennis ) represents a serious threat to North American and European ash species ( Fraxinus spp.). Spread of EAB westwards, from European Russia and Eastern Ukraine, could lead to dramatic consequences for native European ash populations. Early detection is essential for fast and successful eradication of new populations. In this study, we developed a new TaqMan qPCR assay allowing for sensitive and specific detection of EAB. We tested the specificity of the assay against 17 European Agrilus spp., eight buprestid species and nine species belonging to other wood-associated beetle taxa. The qPCR assay provided reliable amplification from samples with DNA concentrations as low as 0.5 picograms per reaction. Moreover, DNA could be amplified from different sample types, such as egg casings, leaves, faeces and bore dust from larval galleries. Robustness of the assay was verified by performing a blind test with four different laboratories. Here we provide a highly specific, robust and sensitive assay which can be used for enhanced surveillance of Agrilus planipennis on the European continent.

Tomato bushy stunt virus (TBSV), the type member of the genus Tombusvirus in the family Tombusviridae is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and Nicotiana benthamiana. However, Arabidopsis thaliana, the well-established model organism in plant biology, genetics and plant–microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in Saccharomyces cerevisiae, a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect Arabidopsis with high efficiency. We demonstrated by confocal and 3D electron microscopy that in Arabidopsis TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new Arabidopsis-TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant–virus interactions in complement to Saccharomyces cerevisiae.

• Previously we have cloned sucrose: fructan-6-fructosyltransferase (6-SFT) from barley (Hordeum vulgare) and proposed that synthesis of fructans in grasses depends on the concerted action of two main enzymes: sucrose: sucrose-1-fructosyltransferase (1-SST), as in other fructan producing plants, and 6-SFT, found only in grasses. • Here we report the cloning of barley 1-SST, verifying the activity of the encoded protein by expression in Pichia pastoris. As expected, the barley 1-SST is homologous to invertases and fructosyltransferases, and in particular to barley 6-SFT. • The gene expression pattern of 1-SST and 6-SFT, along with the corresponding enzyme activities and fructan levels, were investigated in excised barley leaves subjected to a light-dark regime known to sequentially induce fructan accumulation and mobilization. The turnover of transcripts and enzyme activities of 1-SST and 6-SFT was compared, using appropriate inhibitors. • We found the 1-SST transcripts and enzymatic activity respond quickly, being subject to a rapid turnover. By contrast, the 6-SFT transcripts and enzymatic activity were found to be much more stable. The much higher responsiveness of 1-SST to regulatory processes, as compared with 6-SFT, clearly indicates that 1-SST plays the role of the pacemaker enzyme of fructan synthesis in barley leaves.

Glycoside hydrolase family 32 (GH32) harbors hydrolyzing and transglycosylating enzymes that are highly homologous in their primary structure. Eight amino acids dispersed along the sequence correlated with either hydrolase or glycosyltransferase activity. These were mutated in onion vacuolar invertase (acINV) according to the residue in festuca sucrose:sucrose 1-fructosyltransferase (saSST) and vice versa. acINV(W440Y) doubles transferase capacity. Reciprocally, saSST(C223N) and saSST(F362Y) double hydrolysis. SaSST(N425S) shows a hydrolyzing activity three to four times its transferase activity. Interestingly, modeling acINV and saSST according to the 3D structure of crystallized GH32 enzymes indicates that mutations saSST(N425S), acINV(W440Y), and the previously reported acINV(W161Y) reside very close together at the surface in the entrance of the active-site pocket. Residues in- and outside the sucrose-binding box determine hydrolase and transferase capabilities of GH32 enzymes. Modeling suggests that residues dispersed along the sequence identify a location for acceptor-substrate binding in the 3D structure of fructosyltransferases.

Abstract Plasma membrane-resident receptor kinases (RKs) initiate signaling pathways important for plant immunity and development. In Arabidopsis (Arabidopsis thaliana), the receptor for the elicitor-active peptide epitope of bacterial flagellin, flg22, is encoded by FLAGELLIN SENSING2 (FLS2), which promotes plant immunity. Despite its relevance, the molecular components regulating FLS2-mediated signaling remain largely unknown. We show that plasma membrane ARABIDOPSIS-AUTOINHIBITED Ca2+-ATPase (ACA8) forms a complex with FLS2 in planta. ACA8 and its closest homolog ACA10 are required for limiting the growth of virulent bacteria. One of the earliest flg22 responses is the transient increase of cytosolic Ca2+ ions, which is crucial for many of the well-described downstream responses (e.g. generation of reactive oxygen species and the transcriptional activation of defense-associated genes). Mutant aca8 aca10 plants show decreased flg22-induced Ca2+ and reactive oxygen species bursts and exhibit altered transcriptional reprogramming. In particular, mitogen-activated protein kinase-dependent flg22-induced gene expression is elevated, whereas calcium-dependent protein kinase-dependent flg22-induced gene expression is reduced. These results demonstrate that the fine regulation of Ca2+ fluxes across the plasma membrane is critical for the coordination of the downstream microbe-associated molecular pattern responses and suggest a mechanistic link between the FLS2 receptor complex and signaling kinases via the secondary messenger Ca2+. ACA8 also interacts with other RKs such as BRI1 and CLV1 known to regulate plant development, and both aca8 and aca10 mutants show morphological phenotypes, suggesting additional roles for ACA8 and ACA10 in developmental processes. Thus, Ca2+ ATPases appear to represent general regulatory components of RK-mediated signaling pathways.

Plasma membrane-resident receptor kinases (RKs) initiate signaling pathways important for plant immunity and development. In Arabidopsis (Arabidopsis thaliana), the receptor for the elicitor-active peptide epitope of bacterial flagellin, flg22, is encoded by FLAGELLIN SENSING2 (FLS2), which promotes plant immunity. Despite its relevance, the molecular components regulating FLS2-mediated signaling remain largely unknown. We show that plasma membrane ARABIDOPSIS-AUTOINHIBITED Ca(2+)-ATPase (ACA8) forms a complex with FLS2 in planta. ACA8 and its closest homolog ACA10 are required for limiting the growth of virulent bacteria. One of the earliest flg22 responses is the transient increase of cytosolic Ca(2+) ions, which is crucial for many of the well-described downstream responses (e.g. generation of reactive oxygen species and the transcriptional activation of defense-associated genes). Mutant aca8 aca10 plants show decreased flg22-induced Ca(2+) and reactive oxygen species bursts and exhibit altered transcriptional reprogramming. In particular, mitogen-activated protein kinase-dependent flg22-induced gene expression is elevated, whereas calcium-dependent protein kinase-dependent flg22-induced gene expression is reduced. These results demonstrate that the fine regulation of Ca(2+) fluxes across the plasma membrane is critical for the coordination of the downstream microbe-associated molecular pattern responses and suggest a mechanistic link between the FLS2 receptor complex and signaling kinases via the secondary messenger Ca(2+). ACA8 also interacts with other RKs such as BRI1 and CLV1 known to regulate plant development, and both aca8 and aca10 mutants show morphological phenotypes, suggesting additional roles for ACA8 and ACA10 in developmental processes. Thus, Ca(2+) ATPases appear to represent general regulatory components of RK-mediated signaling pathways.