Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant protein in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as a favorite transient protein expression platform in plant science and molecular pharming. Here, we used in-gel digests and mass spectrometry to monitor the migration and topography of 5,040 plant proteins of agroinfiltrated N. benthamiana within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. For instance, this analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor-like kinases. Transient expression of double-tagged candidate proteins confirmed various processing events in vivo. This extensive proteomic dataset can be investigated further and demonstrates that most plant proteins are proteolytically processed and implicates an extensive proteolytic machinery shaping the proteome of agroinfiltrated N. benthamiana.

In addition to jasmonoyl-isoleucine (JA-Ile), a well-established signaling molecule for plant growth and defense, its precursor, cis-12-oxo-phytodienoic acid (OPDA), is thought to possess independent signaling functions. Its perception in vascular plants is still uncharacterized. Several OPDA functions in Arabidopsis were inferred from a mutant that is affected in the function of the OPDA REDUCTASE3 (OPR3), catalyzing the conversion of OPDA within peroxisomes. Recently, opr3 plants were found to accumulate JA-Ile via a cytosolic OPR2-mediated bypass. Given the uncoupling of OPDA and JA biosynthesis in the JA-deficient mutant opr2opr3, potential OPDA signaling was investigated by a transcriptome approach comparing wild type, opr2opr3 and the JA- and OPDA-deficient mutantallene oxide synthase. Dissecting the wound response of seedlings revealed that OPDA lacked a transcriptional signature, and that previously characterized OPDA-response genes were wound-induced independently of OPDA. Exogenous application of OPDA to opr2opr3 seedlings led to JA-Ile formation and signaling even in absence of OPR2 and OPR3 and resulted in activation of sulfur assimilation. These divergent responses to endogenously synthesized and applied OPDA suggest a compartmentalization of endogenous OPDA which was investigated by a trans-organellar complementation approach. OPR3 complemented the opr2opr3 mutant in terms of fertility and wound-induced JA-Ile production irrespective of its subcellular localization. In vitro enzymatic activity of OPR3, however, showed conversion of OPDA and 4,5-didehydro-JA (4,5-ddh-JA), therefore not allowing to conclude which compound is translocated. Dissecting the conversion of either OPDA or 4,5-ddh-JA by OPR2 and OPR1 organelle variants pointed to a strong OPDA compartmentalization supporting its lacking signaling capacity.

Protein engineering using directed evolution and (semi)rational design has emerged as a powerful strategy for optimizing and enhancing enzymes or proteins with desired properties. Integrating artificial intelligence methods has further enhanced and accelerated protein engineering through predictive models developed in data-driven strategies. However, the lack of explainability and interpretability in these models poses challenges. Explainable Artificial Intelligence addresses the interpretability and explainability of machine learning models, providing transparency and insights into predictive processes. Nonetheless, there is a growing need to incorporate explainable techniques in predicting protein properties in machine learning-assisted protein engineering. This work explores incorporating explainable artificial intelligence in predicting protein properties, emphasizing its role in trustworthiness and interpretability. It assesses different machine learning approaches, introduces diverse explainable methodologies, and proposes strategies for seamless integration, improving trust-worthiness. Practical cases demonstrate the explainable model’s effectiveness in identifying DNA binding proteins and optimizing Green Fluorescent Protein brightness. The study highlights the utility of explainable artificial intelligence in advancing computationally assisted protein design, fostering confidence in model reliability.

Aphids are small insects that have developed specialized mouthparts and effector proteins to establish long-term relationships with plants. The peach-potato aphid, Myzus persicae, is a generalist, feeding on many plant species and capable of transmitting numerous pathogens. This study reveals how host-responsive cathepsins B (CathB) in the oral secretions of M. persicae facilitate aphid survival by modulating plant immune responses. Aphid CathB localize to processing bodies (p-bodies) and recruit key immune regulators EDS1, PAD4, and ADR1 to these bodies, suppressing plant defenses. A plant protein, Acd28.9 (Hsp20 family), counteracts this CathB activity and contributes to plant resistance to aphids. These findings highlight a novel role for p-bodies in plant immunity and uncover a plant resistance mechanism to aphid infestation.

Secreted immune proteases Rcr3 and Pip1 of tomato are both inhibited by Avr2 from the fungal plant pathogen Cladosporium fulvumbut only Rcr3 act as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues from tomato Rcr3 required for Cf-2-mediated signalling and bioengineered various proteases to trigger Avr2/Cf-2 dependent immunity. Despite substantial divergences in Rcr3 orthologs from eggplant and tobacco, only minimal alterations were sufficient to trigger Avr2/Cf-2-triggered immune signalling. Tomato Pip1, by contrast, was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signalling. These residues cluster on one side next to the substrate binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues and that both are suboptimal for binding Avr2. This study advances our understanding of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops.

Plant cells experience a variety of mechanical stresses from both internal and external sources, including turgor pressure, mechanical strains arising from heterogeneous growth between neighboring cells, and environmental factors like touch from soil, rain, or wind [1,2]. These stresses serve as signals at the cell-, tissue- and organismal level to coordinate plant growth during development and stress responses [3]. In plants, the physical cell wall-plasma membrane-microtubule continuum is proposed to be integral in transducing mechanical signals from the exterior to intracellular components [4–6]. Cortical microtubules (CMTs) rapidly reorient in response to mechanical stress to align with the maximal tensile stress direction [7,8]. Several studies proposed that CMTs themselves may act as stress sensors; the precise mechanisms involved in the regulation of CMTs and the modes of sensing, however, are still not clearly understood. Here, we show that IQD2 and KLCR1 are enriched at CMTs in proximity to the plasma membrane. IQD2, which is a bona fide microtubule-associated protein, promotes microtubule localization of KLCR1. By combining cross-linking mass spectrometry (XL-MS) and computational modeling with structure-function studies, we present first experimental insights into the composition and structure of IQD2-KLCR1 complexes. Further, we demonstrate that the IQD2-KLCR1 module is a positive regulator of microtubule mechano-responses in pavement cells. Collectively, our work identifies the IQD2-KLCR1 module as novel regulator of mechanostress-mediated CMT reorientation and provides a framework for future mechanistic studies aimed at a functional dissection of mechanotransduction at the plasma membrane-CMT interface during growth and plant morphogenesis.HighlightsIQD2 and KLCR1 localize to the plasma membrane-microtubule nexusIQD2 is required for efficient microtubule targeting of KLCR1in plantaIQD2 physically interacts with KLCR1 and microtubulesThe IQD2-KLCR1 module promotes mechano-stress induced microtubule reorganization

Background  In viticulture, iron (Fe) chlorosis is a common abiotic stress that impairs plant development and leads to yield and quality losses. Under low availability of the metal, the applied N form (nitrate and ammonium) can play a role in promoting or mitigating Fe deficiency stresses. However, the processes involved are not clear in grapevine. Therefore, the aim of this study was to investigate the response of two grapevine rootstocks to the interaction between N forms and Fe uptake. This process was evaluated in a hydroponic experiment using two ungrafted grapevine rootstocks Fercal (Vitis berlandieri x V. vinifera) tolerant to deficiency induced Fe chlorosis and Couderc 3309 (V. riparia x V. rupestris) susceptible to deficiency induced Fe chlorosis. Results  The results could differentiate Fe deficiency effects, N-forms effects, and rootstock effects. Interveinal chlorosis of young leaves appeared earlier on 3309 C from the second week of treatment with NO3−/NH4+ (1:0)/-Fe, while Fercal leaves showed less severe symptoms after four weeks of treatment, corresponding to decreased chlorophyll concentrations lowered by 75% in 3309 C and 57% in Fercal. Ferric chelate reductase (FCR) activity was by trend enhanced under Fe deficiency in Fercal with both N combinations, whereas 3309 C showed an increase in FCR activity under Fe deficiency only with NO3−/NH4+ (1:1) treatment. With the transcriptome analysis, Gene Ontology (GO) revealed multiple biological processes and molecular functions that were significantly regulated in grapevine rootstocks under Fe-deficient conditions, with more genes regulated in Fercal responses, especially when both forms of N were supplied. Furthermore, the expression of genes involved in the auxin and abscisic acid metabolic pathways was markedly increased by the equal supply of both forms of N under Fe deficiency conditions. In addition, changes in the expression of genes related to Fe uptake, regulation, and transport reflected the different responses of the two grapevine rootstocks to different N forms. Conclusions  Results show a clear contribution of N forms to the response of the two grapevine rootstocks under Fe deficiency, highlighting the importance of providing both N forms (nitrate and ammonium) in an appropriate ratio in order to ease the rootstock responses to Fe deficiency.

Background ‘Candidatus Phytoplasma mali’, the causal agent of apple proliferation disease, exerts influence on its host plant through various effector proteins, including SAP11CaPm which interacts with different TEOSINTE BRANCHED1/ CYCLOIDEA/ PROLIFERATING CELL FACTOR 1 and 2 (TCP) transcription factors. This study examines the transcriptional response of the plant upon early expression of SAP11CaPm. For that purpose, leaves of Nicotiana occidentalis H.-M. Wheeler were Agrobacterium-infiltrated to induce transient expression of SAP11CaPm and changes in the transcriptome were recorded until 5 days post infiltration.Results The RNA-seq analysis revealed that presence of SAP11CaPm in leaves leads to downregulation of genes involved in defense response and related to photosynthetic processes, while expression of genes involved in energy production was enhanced.Conclusions The results indicate that early SAP11CaPm expression might be important for the colonization of the host plant since phytoplasmas lack many metabolic genes and are thus dependent on metabolites from their host plant.

Crop protection strategies relying on the improvement of the natural plant immune system via genetic engineering are sustainable solutions against the pathogen thread on food security. Here we describe a novel way to improve the plant immune system by immune protease engineering. As proof of concept, we increased resistance against the late blight pathogen Phytopththora infestans by rendering the tomato secreted immune protease Pip1 insensitive to the P. infestans-secreted inhibitor Epic2B. This concept can be applied to secreted immune proteases in crops by precision breeding.

One class of enzymes that plant pathogens employ to manipulate innate immunity and physiology of the infected cells are host-targeted ADP-ribosyltransferases. The bacterial pathogen Pseudomonas syringae uses its type III secretion system to inject several effector proteins with ADP-ribosyltransferase activity into plant cells. One of them, AvrRpm1, ADP-ribosylates the plasma membrane-associated RPM1-INTERACTING PROTEIN 4 (RIN4) in Glycine max and Arabidopsis thaliana to attenuate targeted secretion of defense-promoting compounds. Substrate identification of host-targeted ADP-ribosyltransferases is complicated by the biochemical lability of the protein modification during plant protein extraction and in several cases required prior knowledge on plant immune signaling pathways that are impaired by the ADP-ribosylating type III effector. Using the AvrRpm1-RIN4 pair as a proof-of-concept, we present an untargeted proteomics workflow for enrichment and detection of ADP-ribosylated proteins and peptides from plant cell extracts that in several cases provides site-resolution for the modification.