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Over the past decade, ample transcriptome data have been generated at different stages during seed germination; however, far less is known about protein synthesis during this important physiological process. Generally, the correlation between transcript levels and protein abundance is low, which strongly limits the use of transcriptome data to accurately estimate protein expression. Polysomal profiling has emerged as a tool to identify mRNAs that are actively translated. The association of the mRNA to the polysome, also referred to as translatome, provides a proxy for mRNA translation. In this study, the correlation between the changes in total mRNA, polysome-associated mRNA, and protein levels across seed germination was investigated. The direct correlation between polysomal mRNA and protein abundance at a single time-point during seed germination is low. However, once the polysomal mRNA of a time-point is compared to the proteome of the next time-point, the correlation is much higher. 35% of the investigated proteome has delayed changes at the protein level. Genes have been classified based on their delayed protein changes, and specific motifs in these genes have been identified. Moreover, mRNA and protein stability and mRNA length have been found as important predictors for changes in protein abundance. In conclusion, polysome association and/or dissociation predicts future changes in protein abundance in germinating seeds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bai, van der Horst, Cordewener, America, Nijveen and Bentsink.)

L-type lectin receptor kinases (LecRK) are potential immune receptors. Here, we characterized two closely-related Arabidopsis LecRK, LecRK-IX.1 and LecRK-IX.2, of which T-DNA insertion mutants showed compromised resistance to Phytophthora brassicae and Phytophthora capsici, with double mutants showing additive susceptibility. Overexpression of LecRK-IX.1 or LecRK-IX.2 in Arabidopsis and transient expression in Nicotiana benthamiana increased Phytophthora resistance but also induced cell death. Phytophthora resistance required both the lectin domain and kinase activity, but for cell death, the lectin domain was not needed. Silencing of the two closely related mitogen-activated protein kinase genes NbSIPK and NbNTF4 in N. benthamiana completely abolished LecRK-IX.1-induced cell death but not Phytophthora resistance. Liquid chromatography-mass spectrometry analysis of protein complexes coimmunoprecipitated in planta with LecRK-IX.1 or LecRK-IX.2 as bait, resulted in the identification of the N. benthamiana ABC transporter NbPDR1 as a potential interactor of both LecRK. The closest homolog of NbPDR1 in Arabidopsis is ABCG40, and coimmunoprecipitation experiments showed that ABCG40 associates with LecRK-IX.1 and LecRK-IX.2 in planta. Similar to the LecRK mutants, ABCG40 mutants showed compromised Phytophthora resistance. This study shows that LecRK-IX.1 and LecRK-IX.2 are Phytophthora resistance components that function independent of each other and independent of the cell-death phenotype. They both interact with the same ABC transporter, suggesting that they exploit similar signal transduction pathways.

Sensitivity of biosensors depends on the orientation of bio-receptors on the sensor surface. The objective of this study was to organize bio-receptors on surfaces in a way that their analyte binding site is exposed to the analyte solution. VHH proteins recognizing foot-and-mouth disease virus (FMDV) were used for making biosensors, and azides were introduced in the VHH to function as bioorthogonal reactive groups. The importance of the orientation of bio-receptors was addressed by comparing sensors with randomly oriented VHH (with multiple exposed azide groups) to sensors with uniformly oriented VHH (with only a single azide group). A surface plasmon resonance (SPR) chip exposing cyclooctyne was reacted to azide functionalized VHH domains, using click chemistry. Comparison between randomly and uniformly oriented bio-receptors showed up to 800-fold increase in biosensor sensitivity. This technique may increase the containment of infectious diseases such as FMDV as its strongly enhanced sensitivity may facilitate early diagnostics., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Plants employ a large number of receptors localizing to the cell surface to sense extracellular signals. Receptor-like proteins (RLPs) form an important group of such trans-membrane receptors, containing an extracellular domain which is involved in signal perception and a short cytoplasmic domain. In contrast to receptor-like kinases (RLKs), RLPs lack a cytoplasmic kinase domain. How intracellular signaling is triggered downstream of RLPs upon perception of an extracellular signal, is therefore still poorly understood. Recently, the RLK SOBIR1 (Suppressor Of BIR1-1) was identified as an essential regulatory RLK of various RLPs involved in plant immunity against fungal pathogens. (1) Given that SOBIR1 appears to be a crucial component of RLP-containing complexes, we aimed to identify additional proteins interacting with SOBIR1. Here, we report on the immunopurification of a functional Arabidopsis thaliana (At)SOBIR1-yellow fluorescent protein (YFP) fusion protein stably expressed in Arabidopsis, followed by mass-spectrometry to identify co-purifying proteins. Interestingly, and in line with various studies showing interaction between RLPs and SOBIR1, we discovered that AtSOBIR1 interacts with AtRLP23, an RLP of which the function is currently unknown.

The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain extracellular leucine-rich repeats and an intracellular kinase domain for activation of downstream signaling, as well as receptor-like proteins (RLPs) that lack this signaling domain. It is therefore hypothesized that RLKs are required for RLPs to activate downstream signaling. The RLPs Cf-4 and Ve1 of tomato (Solanum lycopersicum) mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. Despite their importance, the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. Here we show that the tomato ortholog of the Arabidopsis thaliana RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR) and its close homolog S. lycopersicum (Sl)SOBIR1-like interact in planta with both Cf-4 and Ve1 and are required for the Cf-4- and Ve1-mediated hypersensitive response and immunity. Tomato SOBIR1/EVR interacts with most of the tested RLPs, but not with the RLKs FLS2, SERK1, SERK3a, BAK1, and CLV1. SOBIR1/EVR is required for stability of the Cf-4 and Ve1 receptors, supporting our observation that these RLPs are present in a complex with SOBIR1/EVR in planta. We show that SOBIR1/EVR is essential for RLP-mediated immunity and propose that the protein functions as a regulatory RLK of this type of cell-surface receptors.

Cf proteins are receptor-like proteins (RLPs) that mediate resistance of tomato (Solanum lycopersicum) to the foliar pathogen Cladosporium fulvum. These transmembrane immune receptors, which carry extracellular leucine-rich repeats that are subjected to posttranslational glycosylation, perceive effectors of the pathogen and trigger a defense response that results in plant resistance. To identify proteins required for the functionality of these RLPs, we performed immunopurification of a functional Cf-4-enhanced green fluorescent protein fusion protein transiently expressed in Nicotiana benthamiana, followed by mass spectrometry. The endoplasmic reticulum (ER) heat shock protein70 binding proteins (BiPs) and lectin-type calreticulins (CRTs), which are chaperones involved in ER-quality control, were copurifying with Cf-4-enhanced green fluorescent protein. The tomato and N. benthamiana genomes encode four BiP homologs and silencing experiments revealed that these BiPs are important for overall plant viability. For the three tomato CRTs, virus-induced gene silencing targeting the plant-specific CRT3a gene resulted in a significantly compromised Cf-4-mediated defense response and loss of full resistance to C. fulvum. We show that upon knockdown of CRT3a the Cf-4 protein accumulated, but the pool of Cf-4 protein carrying complex-type N-linked glycans was largely reduced. Together, our study on proteins required for Cf function reveals an important role for the CRT ER chaperone CRT3a in the biogenesis and functionality of this type of RLP involved in plant defense.

Plants lack the seemingly unlimited receptor diversity of a somatic adaptive immune system as found in vertebrates and rely on only a relatively small set of innate immune receptors to resist a myriad of pathogens. Here, we show that disease-resistant tomato plants use an efficient mechanism to leverage the limited nonself recognition capacity of their innate immune system. We found that the extracellular plant immune receptor protein Cf-2 of the red currant tomato (Solanum pimpinellifolium) has acquired dual resistance specificity by sensing perturbations in a common virulence target of two independently evolved effectors of a fungus and a nematode. The Cf-2 protein, originally identified as a monospecific immune receptor for the leaf mold fungus Cladosporium fulvum, also mediates disease resistance to the root parasitic nematode Globodera rostochiensis pathotype Ro1-Mierenbos. The Cf-2-mediated dual resistance is triggered by effector-induced perturbations of the apoplastic Rcr3(pim) protein of S. pimpinellifolium. Binding of the venom allergen-like effector protein Gr-VAP1 of G. rostochiensis to Rcr3(pim) perturbs the active site of this papain-like cysteine protease. In the absence of the Cf-2 receptor, Rcr3(pim) increases the susceptibility of tomato plants to G. rostochiensis, thus showing its role as a virulence target of these nematodes. Furthermore, both nematode infection and transient expression of Gr-VAP1 in tomato plants harboring Cf-2 and Rcr3(pim) trigger a defense-related programmed cell death in plant cells. Our data demonstrate that monitoring host proteins targeted by multiple pathogens broadens the spectrum of disease resistances mediated by single plant immune receptors.

Background: Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MSE was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome., Results: All examined birch species contained several PR-10 genes. In total, 134 unique sequences were recovered. Sequences were attributed to different genes or pseudogenes that were, in turn, ordered into seven subfamilies. Five subfamilies were common to all birch species. Genes of two subfamilies were expressed in pollen, while each birch species expressed a mixture of isoforms with at least four different isoforms. Isoforms that were similar to isoforms with a high IgE-reactivity (Bet v 1a = PR-10.01A01) were abundant in all species except B. lenta, while the hypoallergenic isoform Bet v 1d (= PR-10.01B01) was only found in B. pendula and its closest relatives., Conclusion: Q-TOF LC-MSE allows efficient screening of Bet v 1 isoforms by determining the presence and relative abundance of these isoforms in pollen. B. pendula contains a Bet v 1-mixture in which isoforms with a high and low IgE-reactivity are both abundant. With the possible exception of B. lenta, isoforms identical or very similar to those with a high IgE-reactivity were found in the pollen proteome of all examined birch species. Consequently, these species are also predicted to be allergenic with regard to Bet v 1 related allergies.

EP1, an extracellular protein from carrot (Daucus carota) cell suspensions, has been partially characterized by means of an antiserum and a cDNA clone. In both embryo and suspension cultures different molecular mass EP1 proteins were detected, some of which (31, 32, 52, and 54 kilodaltons) were bound to the cell wall and released into the medium, whereas others (49, 60, and 62 kilodaltons) were more firmly bound to the cell wall and could be extracted with a salt solution. Immunoprecipitation of in vitro translation products revealed a single primary translation product of 45 kilodaltons, suggesting that EP1 heterogeneity is due to differential posttranslational modification. In seedlings organ-specific modification of EP1 proteins was observed, a phenomenon which did not persist in suspension cultures initiated from different seedling organs. In culture EP1 proteins were only found to be associated with vacuolated, nonembryogenic cells, and on these cells they were localized in loosely attached, pectin-containing cell wall material. Purified 52/54 kilodaltons EP1 proteins did not alleviate the inhibitory effect of the glycosylation inhibitor tunicamycin on somatic embryogenesis.