Comprehensive analysis of the membrane phosphoproteome regulated by oligogalacturonides in Arabidopsis thaliana

Early changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity

Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea

Phytoalexins are heterogeneous low molecular mass secondary metabolites with antimicrobial activity produced in response to pathogen invasion attempts at the infection site and represent an important part of the plant defense repertoire. Camalexin (3-Thiazol-2′-yl-indole) is a known phytoalexin first detected and isolated in Camelina sativa, from which it takes its name, infected with Alternaria brassicae (Browne et al., 1991). Production of camalexin is also induced in Arabidopsis thaliana leaves by a range of biotrophic and necrotrophic plant pathogens (bacteria, oomycetes, fungi and viruses) (Ahuja et al., 2012) as well as by abiotic stresses, such as UV and chemicals (e.g. acifluorfen, paraquat, chlorsulfuron and α-amino butyric acid) (Zhao et al., 1998; Tierens et al., 2002). Camalexin originates from tryptophan and CYP79B2 and CYP71B15 (PAD3) are P450 enzymes that catalyze important steps in its biosynthetic pathway (Glawischnig, 2007). In this protocol the detection and quantification of camalexin produced in Arabidopsis leaves infected with the necrotrophic fungus Botrytis cinerea is described

Autologous hematopoietic stem cell transplantation versus low-dose immunosuppression in secondary-progressive multiple sclerosis

BACKGROUND AND PURPOSE: Effectiveness of autologous haematopoietic stem cell transplantation (AHSCT) in relapsing–remitting multiple sclerosis (MS) is well known, but in secondary–progressive (SP)‐MS it is still controversial. Therefore, AHSCT activity was evaluated in SP‐MS using low‐dose immunosuppression with cyclophosphamide (Cy) as a comparative treatment. METHODS: In this retrospective monocentric 1:2 matched study, SP‐MS patients were treated with intermediate‐intensity AHSCT (cases) or intravenous pulses of Cy (controls) at a single academic centre in Florence. Controls were selected according to baseline characteristics adopting cardinality matching after trimming on the estimated propensity score. Kaplan–Meier and Cox analyses were used to estimate survival free from relapses (R‐FS), survival free from disability progression (P‐FS), and no evidence of disease activity 2 (NEDA‐2). RESULTS: A total of 93 SP‐MS patients were included: 31 AHSCT, 62 Cy. Mean follow‐up was 99 months in the AHSCT group and 91 months in the Cy group. R‐FS was higher in AHSCT compared to Cy patients: at Year 5, 100% versus 52%, respectively (p < 0.0001). P‐FS did not differ between the groups (at Year 5: 70% in AHSCT and 81% in Cy, p = 0.572), nor did NEDA‐2 (p = 0.379). A sensitivity analysis including only the 31 “best‐matched” controls confirmed these results. Three neoplasms (2 Cy, 1 AHSCT) and two fatalities (2 Cy) occurred. CONCLUSIONS: This study provides Class III evidence, in SP‐MS, on the superior effectiveness of AHSCT compared to Cy on relapse activity, without differences on disability accrual. Although the suppression of relapses was observed in the AHSCT group only, AHSCT did not show advantages over Cy on disability, suggesting that in SP‐MS disability progression becomes based more on noninflammatory neurodegeneration than on inflammation

Il sistema immunitario della pianta: basi biochimiche e molecolari del riconoscimento dei patogeni e dell'attivazione delle difese della pianta

Durante tutta la loro vita, le piante sono impegnate in una continua lotta con potenziali agenti patogeni. Per essere patogenici, la maggior parte dei microbi deve avere accesso all’interno della pianta, penetrando direttamente attraverso la superficie della foglia o della radice o entrando attraverso ferite o aperture naturali come gli stomi. Nelle fasi iniziali dell’infezione, il patogeno viene contrastato da difese strutturali preformate (ad esempio la parete cellulare della pianta) e biochimiche (ad esempio le fitoanticipine). Inoltre, le cellule vegetali sono in grado di riconoscere rapidamente un microrganismo potenzialmente pericoloso e attivare una vasta gamma di risposte volte a ucciderlo e a limitarne la diffusione nell’ospite. È importante sottolineare che le piante discriminano tra patogeni e microbi innocui o benefici, assicurando che adeguate risposte, che sono costose per la pianta, siano indotte solo quando necessarie. Le piante non hanno cellule immunitarie circolanti e non hanno un sistema immunitario adattativo come nei vertebrati; invece, ogni cellula è in grado di riconoscere la presenza di un agente patogeno, attraverso meccanismi biochimici autonomi che assomigliano al sistema immunitario innato degli animali. Per questo motivo, parliamo spesso della immunità innata delle piante per descrivere il complesso di risposte di difesa che vengono rapidamente indotte nel sito di infezione. Successivamente, si possono osservare risposte più a lungo termine non solo nei tessuti direttamente a contatto con l’agente patogeno, ma anche nel resto della pianta, e può verificarsi una resistenza acquisita alle infezioni successive

Large-scale proteome analysis of tomato fruit microsomes

Solanum lycopersicum (tomato) is one of the most important plant crops and a model system to study development and ripening of fleshy fruit. The membrane system is very important for all biological processes; for example, the endoplasmic reticulum (ER) and the Golgi apparatus play a pivotal role in the secretion of proteins and in the synthesis of the non-cellulosic portion of the cell wall. The aim of this work was to characterize the membrane proteome profile of tomato fruit at the ‘‘mature green’’ ripening stage. Total microsomes were prepared and separated by centrifugation through a iodixanol continuous gradient. The organelle distribution pattern (plasma membrane, ER, Golgi, chloroplast, nucleus) was determined using known markers of these compartments. Proteins were identified using a combination of 1-D SDS polyacrylamide gel electrophoresis and nanoLC-ESIMS/ MS. After electrophoresis, each lane was cut into 14 pieces, digested with trypsin and analyzed by nano-HPLC coupled to an Orbitrap mass spectrometer. More than 2400 different proteins were identified, for which the Arabidopsis homologue was searched and subjected to GO analysis to determine the represented biological process, molecular function and subcellular localization. GO term enrichment analysis confirmed the enrichment in membrane proteins. This large scale proteomic analysis provides a detailed reference map of the membrane proteome at specific stages of tomato fruit development and a background for comparison of physiological processes such as ripening or biotic and abiotic stresses

Ruolo degli ormoni vegetali nella regolazione delle risposte immunitarie

Essendo organismi sessili, le piante monitorano continuamente l’ambiente che le circonda e modificano, di conseguenza, la loro crescita, sviluppo e difesa al fine di adattarsi e ottimizzare la produttività. Le piante hanno spesso necessità di contrastare i tentativi di invasione di diversi agenti patogeni microbici: virus, batteri, funghi e oomiceti. Sulla base delle diverse strategie di infezione, i fitopatogeni possono essere classificati come biotrofici o necrotrofici. I patogeni biotrofici si moltiplicano dapprima negli spazi intercellulari nutrendosi di cellule vegetali viventi, mentre i patogeni necrotrofici uccidono le cellule ospiti utilizzando metaboliti tossici e quindi ottengono i nutrienti dalle cellule morte. Durante la coevoluzione con gli agenti patogeni, le piante hanno sviluppato sofisticati sistemi di percezione e sistemi di difesa inducibili per limitarne l’invasione. Gli ormoni vegetali, o fitormoni, sono piccole molecole di segnalazione endogene con diverse proprietà chimiche che svolgono ruoli critici nell’adattamento ai cambiamenti ambientali e governano le risposte a un’ampia varietà di stress biotici e abiotici regolando l’equilibrio tra crescita delle piante e risposta di difesa. L’attività di un determinato ormone dipende dalla sua biosintesi, coniugazione, trasporto a lunga e/o breve distanza, degradazione nonché attivazione e inattivazione ormonale. Gli ormoni vegetali comprendono etilene (ET), acido jasmonico (JA) e acido salicilico (SA) che svolgono un ruolo centrale nella regolazione delle risposte immunitarie delle piante. Inoltre, anche per altri ormoni vegetali, come l’auxina (acido indol- 3-acetico, IAA), l’acido abscissico (ABA), le citokinine, le gibberelline e i brassinosteroidi (BR), ben noti per il loro ruolo nella regolazione dello sviluppo e della crescita delle piante, è emerso recentemente un ruolo come regolatori chiave dell’immunità delle piante. Gli ormoni vegetali interagiscono tra di loro in complessi network per bilanciare la risposta a segnali di sviluppo e ambientali e quindi limitare i costi di fitness associati alla difesa. I meccanismi molecolari che regolano questi network di segnalazione ormonali sono in gran parte sconosciuti. Inoltre, i patogeni mirano alle vie di segnalazione ormonale per disturbare ed eludere le risposte di difesa delle piante

Phosphoproteomics of early oligogalacturonides signaling in Arabidopsis

During the infection of a plant tissue by phytopathogenic fungi, homogalacturonan, the main component of pectin, is broken down into fragments called oligogalacturonides (OGs) by the action of fungal polygalacturonases. The interaction between polygalacturonases and their inhibitors (polygalacturonase-inhibiting proteins: PGIPs) in the plant cell wall favours the formation of OGs with degree of polymerization from 10 to 15 that function as signals for the activation of the plant innate immune response. Although the effects of OGs in plant defence are well recognised, the perception/transduction mechanisms of these elicitors are still not completely described. By a 2D-DIGE approach coupled with the quantitative phospho-specific stain ProQ Diamond we studied the early events of phosphorylation in Arabidopsis thaliana in response to OGs. Soluble proteins and prefractionated total membranes were analyzed and the differential phosphorylated proteins were identified by MALDI-ToF mass spectrometry. Phosphorylation changes were detected in several oxidative stress-related proteins that included heat shock proteins, jasmonate-inducible jacalin proteins and H+-ATPases, among which the vacuolar-H+ATPase encoded by DET3 gene. Phosphorilation changes were also detected in plasma membrane proteins like AtPcaP1, a plasma membrane-bounded protein involved in the cross-talk between Ca+2 signaling and PtdInsPs in the intracellular signaling pathway

Polygalacturonases, polygalacturonase-inhibiting proteins and pectic oligomers in plant-pathogen interactions

Polygalacturonases (PGs) are produced by fungal pathogens during early plant infection and are believed to be important pathogenicity factors. Polygalacturonase-inhibiting proteins (PGIPs) are plant defense proteins which reduce the hydrolytic activity of endoPGs and favor the accumulation of long-chain oligogalacturonides (OGs) which are elicitors of a variety of defense responses. PGIPs belong to the superfamily of leucine reach repeat (LRR) proteins which also include the products of several plant resistance genes. A number of evidence demonstrates that PGIPs efficiently inhibit fungal invasion

Aqueous solution properties of bacterial poly-gamma-D-glutamate

A preliminary physico-chemical characterization of a bacterial poly-γ-glutamate sample (96% D-glutamic acid content), γ-D-PGA, in dilute aqueous solutions has been carried out by means of potentiometric, viscosimetric, infrared and chiroptical spectroscopic experiments. The biopolymer exhibits properties strikingly dependent on a number of parameters, mainly: polymer concentration, pH, ionic strength, and nature of added salt. In dilute solutions (polymer concentration around 0.1% w/V) and for pH > 7, γ-D-PGA chains assume elongated, stiff conformations while upon protonation (pH < 3) globular states would prevail. Addition of divalent counterions (Ca(II)) also leads to compact γ-D-PGA conformations