Unveiling orphan receptor-like kinases in plants: novel client discovery using high-confidence library predictions in the Kinase-Client (KiC) assay.

Plants are remarkable in their ability to adapt to changing environments, with receptor-like kinases (RLKs) playing a pivotal role in perceiving and transmitting environmental cues into cellular responses. Despite extensive research on RLKs from the plant kingdom, the function and activity of many kinases, i.e., their substrates or "clients", remain uncharted. To validate a novel client prediction workflow and learn more about an important RLK, this study focuses on P2K1 (DORN1), which acts as a receptor for extracellular ATP (eATP), playing a crucial role in plant stress resistance and immunity. We designed a Kinase-Client (KiC) assay library of 225 synthetic peptides, incorporating previously identified P2K phosphorylated peptides and novel predictions from a deep-learning phosphorylation site prediction model (MUsite) and a trained hidden Markov model (HMM) based tool, HMMER. Screening the library against purified P2K1 cytosolic domain (CD), we identified 46 putative substrates, including 34 novel clients, 27 of which may be novel peptides, not previously identified experimentally. Gene Ontology (GO) analysis among phosphopeptide candidates revealed proteins associated with important biological processes in metabolism, structure development, and response to stress, as well as molecular functions of kinase activity, catalytic activity, and transferase activity. We offer selection criteria for efficient further in vivo experiments to confirm these discoveries. This approach not only expands our knowledge of P2K1's substrates and functions but also highlights effective prediction algorithms for identifying additional potential substrates. Overall, the results support use of the KiC assay as a valuable tool in unraveling the complexities of plant phosphorylation and provide a foundation for predicting the phosphorylation landscape of plant species based on peptide library results.

The Raf-like MAPKKK INTEGRIN-LINKED KINASE 5 regulates purinergic receptor-mediated innate immunity in Arabidopsis.

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in eukaryotic defense against various pathogens. Activation of the extracellular ATP (eATP) receptor P2K1 triggers MAP kinase 3 and 6 (MPK3/6) phosphorylation, which leads to an elevated plant defense response. However, the mechanism by which P2K1 activates the MAPK cascade is unclear. In this study, we show that in Arabidopsis thaliana, P2K1 phosphorylates the Raf-like MAP kinase kinase kinase (MAPKKK) INTEGRIN-LINKED KINASE 5 (ILK5) on serine 192 in the presence of eATP. The interaction between P2K1 and ILK5 was confirmed both in vitro and in planta and their interaction was enhanced by ATP treatment. Similar to P2K1 expression, ILK5 expression levels were highly induced by treatment with ATP, flg22, Pseudomonas syringae pv. tomato DC3000, and various abiotic stresses. ILK5 interacts with and phosphorylates the MAP kinase MKK5. Moreover, phosphorylation of MPK3/6 was significantly reduced upon ATP treatment in ilk5 mutant plants, relative to wild-type (WT). The ilk5 mutant plants showed higher susceptibility to P. syringae pathogen infection relative to WT plants. Plants expressing only the mutant ILK5S192A protein, with decreased kinase activity, did not activate the MAPK cascade upon ATP addition. These results suggest that eATP activation of P2K1 results in transphosphorylation of the Raf-like MAPKKK ILK5, which subsequently triggers the MAPK cascade, culminating in activation of MPK3/6 associated with an elevated innate immune response.

A survey of Methylobacterium species and strains reveals widespread production and varying profiles of cytokinin phytohormones.

BACKGROUND: Symbiotic Methylobacterium strains comprise a significant part of plant microbiomes. Their presence enhances plant productivity and stress resistance, prompting classification of these strains as plant growth-promoting bacteria (PGPB). Methylobacteria can synthesize unusually high levels of plant hormones, called cytokinins (CKs), including the most active form, trans-Zeatin (tZ). RESULTS: This study provides a comprehensive inventory of 46 representatives of Methylobacterium genus with respect to phytohormone production in vitro, including 16 CK forms, abscisic acid (ABA) and indole-3-acetic acid (IAA). High performance-liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analyses revealed varying abilities of Methylobacterium strains to secrete phytohormones that ranged from 5.09 to 191.47 pmol mL-1 for total CKs, and 0.46 to 82.16 pmol mL-1 for tZ. Results indicate that reduced methanol availability, the sole carbon source for bacteria in the medium, stimulates CK secretion by Methylobacterium. Additionally, select strains were able to transform L-tryptophan into IAA while no ABA production was detected. CONCLUSIONS: To better understand features of CKs in plants, this study uncovers CK profiles of Methylobacterium that are instrumental in microbe selection for effective biofertilizer formulations.

Using Proteomic Approaches to Unravel the Response of Ctenocephalides felis felis to Blood Feeding and Infection With Bartonella henselae.

Among the Ctenocephalides felis felis-borne pathogens, Bartonella henselae, the main aetiological agent of cat scratch disease (CSD), is of increasing comparative biomedical importance. Despite the importance of B. henselae as an emergent pathogen, prevention of the diseases caused by this agent in cats, dogs and humans mostly relies on the use of ectoparasiticides. A vaccine targeting both flea fitness and pathogen competence is an attractive choice requiring the identification of flea proteins/metabolites with a dual effect. Even though recent developments in vector and pathogen -omics have advanced the understanding of the genetic factors and molecular pathways involved at the tick-pathogen interface, leading to discovery of candidate protective antigens, only a few studies have focused on the interaction between fleas and flea-borne pathogens. Taking into account the period of time needed for B. henselae replication in flea digestive tract, the present study investigated flea-differentially abundant proteins (FDAP) in unfed fleas, fleas fed on uninfected cats, and fleas fed on B. henselae-infected cats at 24 hours and 9 days after the beginning of blood feeding. Proteomics approaches were designed and implemented to interrogate differentially expressed proteins, so as to gain a better understanding of proteomic changes associated with the initial B. henselae transmission period (24 hour timepoint) and a subsequent time point 9 days after blood ingestion and flea infection. As a result, serine proteases, ribosomal proteins, proteasome subunit α-type, juvenile hormone epoxide hydrolase 1, vitellogenin C, allantoinase, phosphoenolpyruvate carboxykinase, succinic semialdehyde dehydrogenase, glycinamide ribotide transformylase, secreted salivary acid phosphatase had high abundance in response of C. felis blood feeding and/or infection by B. henselae. In contrast, high abundance of serpin-1, arginine kinase, ribosomal proteins, peritrophin-like protein, and FS-H/FSI antigen family member 3 was strongly associated with unfed cat fleas. Findings from this study provide insights into proteomic response of cat fleas to B. henselae infected and uninfected blood meal, as well as C. felis response to invading B. henselae over an infection time course, thus helping understand the complex interactions between cat fleas and B. henselae at protein levels.

Identification of novel protein-coding sequences in Eucalyptus grandis plants by high-resolution mass spectrometry.

Eucalyptus species are widely used in the forestry industry, and a significant increase in the number of sequences available in database repositories has been observed for these species. In proteomics, a protein is identified by correlating the theoretical fragmentation spectrum derived from genomic/transcriptomic data against the experimental fragmentation mass spectrum acquired from large-scale analysis of protein mixtures. Proteogenomics is an alternative approach that can identify novel proteins encoded by regions previously considered as non-coding. This study aimed to confidently identify and confirm the existence of previously unknown protein-coding sequences in the Eucalyptus grandis genome. To this end, we used a modified spectral correlation strategy and a dedicated de novo peptide sequencing pipeline. Upon the strategy used here, we confidently identified 41 novel peptide forms and six peptides containing at least one single amino acid substitution. The most representative genomic class of novel peptides was identified as originating from alternative reading frames. In contrast, no clear single amino acid substitution pattern was identified. Validation of the identifications was carried out using a parallel reaction monitoring approach that provided further mass spectrometry support for the existence of the novel peptide sequences. Data are available via ProteomeXchange with identifier PXD022110.

Agronomic performance and genetic diversity among lines and soybean cultivars / Desempenho agronômico e diversidade genética em linhagens e cultivares de soja

Knowledge of genetic diversity is of great importance to the contribution of plant breeding, as it helps plant breeders to better selecting parents for establishment of segregating populations. This knowledge is especially important to crops that presents a narrow genetic base as soybean. The objective of this work was to assess agronomic performance and genetic diversity of lines and soybean cultivars, in order to find those ones with potential to be used as parents in breeding programs. Experiment was carried out during 2015/2016 season in the Federal University of Uberlândia farm known as Capim Branco. A randomized complete block designed was applied in order to assess twentythree morpho-agronomic features on twenty-four soybean genotypes. Significant genetic variability for all agronomic traits was found. Genotypes UFU B7, UFU B14, UFU B16, Msoy 6101 and UFUS 7910 were set as early maturity soybean, with life cycle duration ranging from 107.3 to 111 days. Genotypes UFU B1, UFU B3, UFU B8 and UFU B16 presented productivity values higher than national average of 2.870 kg ha-1. Genotypes were clustered by UPGMA, Tocher and canonical variables methods. Both UPGMA and Tocher methods formed four groups. NDF was the major contributor to genetic diversity. Hybridizations between UFU B3, UFU B8, UFU B16 genotypes with UFU B1 genotype might be promising for establishment of segregating populations shown superior genetic variability. Under these circumstances, it was concluded that agronomic features assessed were useful on detection of parents with potential to UFU's soybean breeding program.

O conhecimento da diversidade genética é de grande importância para o melhoramento de plantas, pois auxiliam os melhoristas na escolha de genitores para formação de populações segregantes, especialmente na cultura da soja que é de base genética estreita. O objetivo do trabalho foi avaliar o desempenho agronômico e a diversidade genética de linhagens e cultivares de soja, com potencial de serem utilizados como genitores em programas de melhoramento genético. O experimento foi conduzido em uma área experimental localizada na Fazenda Capim Branco pertencente à Universidade Federal de Uberlândia na safra de 2015/2016. Foram avaliados 24 genótipos de soja, em delineamento de blocos completos casualizados, nos quais foram avaliados 23 caracteres morfo-agronômicos. Foi verificada variabilidade genética para todos os caracteres agronômicos. Os genótipos UFU B7, UFU B14, UFU B16, Msoy 6101 e UFUS 7910 foram classificados como genótipos de ciclo precoce, com duração variando de 107,3 a 111 dias. Os genótipos UFU B1, UFU B3, UFU B8 e UFU B16 apresentaram produtividade elevadas em relação aos demais com valores superiores à média nacional, que é de 2.870 kg ha-1. Os genótipos foram agrupados pelos métodos UPGMA, Tocher e variáveis canônicas. Pelos métodos UPGMA e Tocher foram formados quatro grupos distintos. O NDF foi o caractere que mais contribuiu com a diversidade genética. Hibridações dos genótipos UFU B3, UFU B8 e UFU B16 com o genótipo UFU B1 são promissoras para obtenção de populações segregantes com variabilidade genética superior. Desse modo concluiu-se que o uso de caracteres agronômicos permitiu detectar genitores potenciais para o programa de melhoramento de soja da UFU.