Discovering the RNA-binding proteome of plant leaves with an improved RNA interactome capture method

RNA-binding proteins (RBPs) play a crucial role in regulating RNA function and fate. However, the full complement of RBPs has only recently begun to be uncovered through proteome-wide approaches such as RNA interactome capture (RIC). RIC has been applied to various cell lines and organisms, including plants, greatly expanding the repertoire of RBPs. However, several technical challenges have limited the efficacy of RIC when applied to plant tissues. Here, we report an improved version of RIC that overcomes the difficulties imposed by leaf tissue. Using this improved RIC method in Arabidopsis leaves, we identified 717 RBPs, generating a deep RNA-binding proteome for leaf tissues. While 75% of these RBPs can be linked to RNA biology, the remaining 25% were previously not known to interact with RNA. Interestingly, we observed that a large number of proteins related to photosynthesis associate with RNA in vivo, including proteins from the four major photosynthetic supercomplexes. As has previously been reported for mammals, a large proportion of leaf RBPs lack known RNA-binding domains, suggesting unconventional modes of RNA binding. We anticipate that this improved RIC method will provide critical insights into RNA metabolism in plants, including how cellular RBPs respond to environmental, physiological and pathological cues

Extractive Latifundio and the Low Income of the Extractive Reserve Rio Ouro Preto, Western Amazon

In an attempt to mitigate the environmental impacts caused by large farmers, extractive reserves (RESEXs) were founded with the objectives of conserving biological diversity and meeting traditional communities’ vital needs. This study aimed to evaluate whether the objectives of human needs satisfaction and environmental conservation were achieved based on institutional investments in a RESEX in the Brazilian Amazon. The study was developed through the association method, and we confirmed interference due to the dependence relationship that occurred between the variables of the economic and social groups with the institutional one. Here, we conclude that low investments in social (education and health) and productive policies (roads, transport, and technical assistance) prevent social development and consequently motivate deforestation when families seek to ensure subsistence

Extracellular proteolytic cascade in tomato activates immune protease Rcr3

Proteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogen Cladosporium fulvum (syn. Passalora fulva) Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Asp-selective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 of P. infestans can block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation in Nicotiana benthamiana requires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity

Consistent effects of independent domestication events on the plant microbiota

The effect of plant domestication on plant-microbe interactions remains difficult to prove. In this study, we provide evidence of a domestication effect on the composition and abundance of the plant microbiota. We focused on the genus Phaseolus, which underwent four independent domestication events within two species (P. vulgaris and P. lunatus), providing multiple replicates of a process spanning thousands of years. We targeted Phaseolus seeds to identify a link between domesticated traits and bacterial community composition as Phaseolus seeds have been subject to large and consistent phenotypic changes during these independent domestication events. The seed bacterial communities of representative plant accessions from subpopulations descended from each domestication event were analyzed under controlled and field conditions. The results showed that independent domestication events led to similar seed bacterial community signatures in independently domesticated plant populations, which could be partially explained by selection for common domesticated plant phenotypes. Our results therefore provide evidence of a consistent effect of plant domestication on seed microbial community composition and abundance and offer avenues for applying knowledge of the impact of plant domestication on the plant microbiota to improve microbial applications in agriculture

Investigation of Association between PFO Complicated by Cryptogenic Stroke and a Common Variant of the Cardiac Transcription Factor GATA4

Patent foramen ovale (PFO) is associated with clinical conditions including cryptogenic stroke, migraine and varicose veins. Data from studies in humans and mouse suggest that PFO and the secundum form of atrial septal defect (ASDII) exist in an anatomical continuum of septal dysmorphogenesis with a common genetic basis. Mutations in multiple members of the evolutionarily conserved cardiac transcription factor network, including GATA4, cause or predispose to ASDII and PFO. Here, we assessed whether the most prevalent variant of the GATA4 gene, S377G, was significantly associated with PFO or ASD. Our analysis of world indigenous populations showed that GATA4 S377G was largely Caucasian-specific, and so subjects were restricted to those of Caucasian descent. To select for patients with larger PFO, we limited our analysis to those with cryptogenic stroke in which PFO was a subsequent finding. In an initial study of Australian subjects, we observed a weak association between GATA4 S377G and PFO/Stroke relative to Caucasian controls in whom ASD and PFO had been excluded (OR = 2.16; p = 0.02). However, in a follow up study of German Caucasians no association was found with either PFO or ASD. Analysis of combined Australian and German data confirmed the lack of a significant association. Thus, the common GATA4 variant S377G is likely to be relatively benign in terms of its participation in CHD and PFO/Stroke

Intravenous alteplase for stroke with unknown time of onset guided by advanced imaging: systematic review and meta-analysis of individual patient data

Background: Patients who have had a stroke with unknown time of onset have been previously excluded from thrombolysis. We aimed to establish whether intravenous alteplase is safe and effective in such patients when salvageable tissue has been identified with imaging biomarkers. Methods: We did a systematic review and meta-analysis of individual patient data for trials published before Sept 21, 2020. Randomised trials of intravenous alteplase versus standard of care or placebo in adults with stroke with unknown time of onset with perfusion-diffusion MRI, perfusion CT, or MRI with diffusion weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch were eligible. The primary outcome was favourable functional outcome (score of 0–1 on the modified Rankin Scale [mRS]) at 90 days indicating no disability using an unconditional mixed-effect logistic-regression model fitted to estimate the treatment effect. Secondary outcomes were mRS shift towards a better functional outcome and independent outcome (mRS 0–2) at 90 days. Safety outcomes included death, severe disability or death (mRS score 4–6), and symptomatic intracranial haemorrhage. This study is registered with PROSPERO, CRD42020166903. Findings: Of 249 identified abstracts, four trials met our eligibility criteria for inclusion: WAKE-UP, EXTEND, THAWS, and ECASS-4. The four trials provided individual patient data for 843 individuals, of whom 429 (51%) were assigned to alteplase and 414 (49%) to placebo or standard care. A favourable outcome occurred in 199 (47%) of 420 patients with alteplase and in 160 (39%) of 409 patients among controls (adjusted odds ratio [OR] 1·49 [95% CI 1·10–2·03]; p=0·011), with low heterogeneity across studies (I2=27%). Alteplase was associated with a significant shift towards better functional outcome (adjusted common OR 1·38 [95% CI 1·05–1·80]; p=0·019), and a higher odds of independent outcome (adjusted OR 1·50 [1·06–2·12]; p=0·022). In the alteplase group, 90 (21%) patients were severely disabled or died (mRS score 4–6), compared with 102 (25%) patients in the control group (adjusted OR 0·76 [0·52–1·11]; p=0·15). 27 (6%) patients died in the alteplase group and 14 (3%) patients died among controls (adjusted OR 2·06 [1·03–4·09]; p=0·040). The prevalence of symptomatic intracranial haemorrhage was higher in the alteplase group than among controls (11 [3%] vs two [<1%], adjusted OR 5·58 [1·22–25·50]; p=0·024). Interpretation: In patients who have had a stroke with unknown time of onset with a DWI-FLAIR or perfusion mismatch, intravenous alteplase resulted in better functional outcome at 90 days than placebo or standard care. A net benefit was observed for all functional outcomes despite an increased risk of symptomatic intracranial haemorrhage. Although there were more deaths with alteplase than placebo, there were fewer cases of severe disability or death. Funding: None

Identifying apoplastic plant proteins involved in plant-pathogen interactions on the basis of their evolutionary profile

To sustainably feed a growing global population, yield can be increased by reducing crop losses to plant pathogens. During infection of plants, several pathogens attempt to colonise the intercellular space, the apoplast. Apoplastic defence against attempted invasions includes the secretion of defence-related proteins, turning the apoplast into a molecular battlefield. While few apoplastic hydrolases have been characterised in plant immunity, many secreted proteins remain uncharacterised. Thus, in order to improve our understanding of apoplastic plant defence responses, we aimed to identify novel apoplastic proteins involved in immunity in this thesis. We combined comparative proteomics with molecular evolution and bioluminescence-based high-throughput disease assays. First, to identify core extracellular tomato proteins in immune responses, we studied apoplastic fluids (AF) of different biotic stress-induced tomato species S.lycopersicum, S.peruvianum and S.pennellii by mass spectrometry (MS). We identified that P69 subtilases were the most abundant proteins in tomato AFs during defence. Molecular evolution studies further implicated P69 subtilases in plant immunity and implicated another 20 biotic stress-induced tomato proteins under positive selection in plant immunity. Second, we compared apoplastic proteomic landscapes between tomato species and N.benthamiana, G.max and A.thaliana which marked distinct differences in profiles of accumulating defence proteins, implicating subtilases from G.max and chitinases from tomato species in plant immunity. Third, we established a bioluminescence-based, high-throughput disease assay for the N.benthamiana-Pseudomonas syringae pv. tomato (Pto) pathosystem, but failed to identify increased immunity in N.benthamiana against Pto for three proteins implicated in plant immunity, including P69B. Last, we used AlphaFold2 multimer as a high-throughput in silico screening tool to identify inhibitor-enzyme complexes from the plant-pathogen interface and identified 13 novel, putative hydrolase inhibitors, including six putative P69B inhibitors. This thesis has contributed to understanding apoplastic defence responses by identifying proteins implicated in plant immunity

AlphaFold-Multimer predicts cross-kingdom interactions at the plant-pathogen interface

Abstract Adapted plant pathogens from various microbial kingdoms produce hundreds of unrelated small secreted proteins (SSPs) with elusive roles. Here, we used AlphaFold-Multimer (AFM) to screen 1879 SSPs of seven tomato pathogens for interacting with six defence-related hydrolases of tomato. This screen of 11,274 protein pairs identified 15 non-annotated SSPs that are predicted to obstruct the active site of chitinases and proteases with an intrinsic fold. Four SSPs were experimentally verified to be inhibitors of pathogenesis-related subtilase P69B, including extracellular protein-36 (Ecp36) and secreted-into-xylem-15 (Six15) of the fungal pathogens Cladosporium fulvum and Fusarium oxysporum, respectively. Together with a P69B inhibitor from the bacterial pathogen Xanthomonas perforans and Kazal-like inhibitors of the oomycete pathogen Phytophthora infestans, P69B emerges as an effector hub targeted by different microbial kingdoms, consistent with a diversification of P69B orthologs and paralogs. This study demonstrates the power of artificial intelligence to predict cross-kingdom interactions at the plant-pathogen interface