Toward uncovering natural resistance to aphids in plants: study of proteins interacting with Brevicoryne brassicae and Macrosiphum euphorbiae effectors

Riassunto: Gli afidi rappresentano uno dei tipi di parassita più insidiosi in agricoltura e orticultura giacché causano sia danni diretti alle piante, succhiandone la linfa, sia indiretti agendo come vettori di virus fitopatogeni. Alcuni di essi mostrano specificità per l’ospite e si nutrono solamente di una specie in un singolo genere di piante: è il caso di Brevicoryne brassicae, l’afide ceroso delle crucifere, uno dei parassiti più devastanti delle Brassicaceae in tutto il mondo. Altri afidi, come Macrosiphum euphorbiae, l’afide delle patate, hanno un ampio range di piante ospiti e sono capaci di nutrirsi con una grande varietà di piante appartenenti a differenti generi e famiglie. Il controllo di questi parassiti è principalmente basato sul trattamento con insetticidi, ma la crescente preoccupazione sulla dipendenza dei coltivatori dai prodotti chimici e il rischio di contaminazione sia dell’ambiente che degli organismi non target ha sollevato la necessità di utilizzare misure di controllo alternative come l’uso di cultivar resistenti. I grandi progressi fatti nell'ambito della ricerca sulle secrezioni salivari degli afidi, aprono la strada all'uso degli effettori degli afidi (proteine o piccole molecole secrete dai parassiti delle piante allo scopo di alterare la struttura e la funzione delle cellule dell’ospite e di favorire la colonizzazione della pianta) negli studi finalizzati a rivelare i dettagli dell’associazione fra gli insetti fitofagi e i loro ospiti e a scoprire geni candidati che conferiscono resistenza contro i parassiti che si nutrono succhiando la linfa delle piante. Questo studio sperimentale è stato svolto presso l’azienda KeyGene N.V. con sede a Wageningen, Paesi Bassi ed è parte di un progetto finalizzato alla scoperta dei tratti genetici coinvolti nella resistenza agli afidi in pianta. Lo scopo di questo lavoro è quello di individuare in piante modello una serie di geni candidati che conferiscono resistenza a B. brassicae e a M. euphorbiae in modo da introdurre tali tratti di interesse tramite le convenzionali tecniche di breeding in colture importanti dal punto di vista economico come B. napus, pomodoro e peperone. Sono stati utilizzati due approcci differenti per le due specie di afidi prese in considerazione. Per B. brassicae è stato usato come “bait” (esca) per un “pull-down assay” in pianta, l’effettore C002 (una proteina dalla funzione ignota, secreta tramite la saliva che ha dimostrato avere un ruolo essenziale nell’interazione afide-pianta in differenti specie di afidi). La proteina BbC002 fusa con una specifica sequenza peptidica, detta tag, è stata espressa in piante di Arabidopsis: i possibili complessi formati dal legame fra BbC002 e le proteine vegetali sono stati estratti, purificati e identificati tramite spettroscopia di massa. Sono state testate linee mutanti di Arabidopsis per tali proteine target di C002 al fine di identificare eventuali mutanti knock-out che mostrassero una minore o maggiore suscettibilità ad infezione da parte di B. brassicae: il gene o i geni responsabili del cambiamento dell’interazione Arabidopsis-B.brassicae possono essere utilizzare per identificare geni omologhi in altre specie di interesse economico. I possibili interattori vegetali che legano gli effettori di M. euphorbiae, sono stati studiati tramite il saggio del doppio ibrido in lievito: tre effettori dell’afide delle patate, MeC002 e altre due proteine che hanno dimostrato di aumentare la fecondità degli afidi, sono stati testati con una libreria di sequenze di cDNA di peperone. Le sequenze dei tre effettori esaminati sono state isolate dal cDNA dell’afide delle patate e clonate nel vettore di destinazione contenente il dominio di legame di GAL4 per eseguire il saggio del doppio ibrido. Il test per le interazioni positive è stato effettuato trasformando cellule di lievito con due dei tre bait creati e i differenti “prey” (preda) formati dai geni presenti in una libreria normalizzata di cDNA di peperone. English abstract: Aphids are one of the major pests of temperate agricultural and horticultural crops, causing damage either directly by feeding or indirectly by transmitting plant viruses. Some of them are host-specific feeding only on one species in a single host genus: this is the case of B. brassicae, the cabbage aphid, that is one of the most serious pests of brassicas crops throughout the world. Other aphids, such as M. euphorbiae, have a broad range of host plants and are able to feed on a varieties of plants belonging to different genus and families. The control of these pests is primary based on treatments with insecticides but growing concern over the dependence of farmers on agrochemicals and the risk of contamination of the environment and non target organisms has increased the necessity of alternative control measures as the use of resistant cultivars. The increasing interest and the immense progress made in the aphid salivary secretions research field, open the door to the use of aphid effectors (proteins and small molecules secreted by plant pests for the purpose of altering host-cell structure and function and establishing “colonization” of the plant) to reveal the details of the intimate associations between the herbivore insects and their host plants as well as to discover candidate genes that convey tangible resistance against sap sucking pests. This experimental study was developed in the company Keygene N.V., based in Wageningen, The Netherlands and is part of an ongoing project focused on discovering genetic traits involved in aphid resistance in crops. The goal of this work is to investigate in model plants a list of candidate genes that confer plant resistance to B. brassicae and M. euphorbiae in order to introgress the traits of interest through conventional breeding in economic important crops as B. napus, tomato, pepper. Two different approaches were assessed for the two aphids species taken in account. For B. brassicae, the effector C002 (a protein secreted by aphid saliva with unknown function that showed to play an essential role in aphid-plant interaction in different species of aphids) was used as a bait protein for an in planta pull-down experiment. BbC002 protein fused with a specific peptide sequence (tag) was expressed in Arabidopsis plants: the possible complexes formed from BbC002:plant protein binding were extracted from plants, purified and identified through mass spectrometry analysis. Once characterized C002 targets, Arabidopsis mutants lines of these proteins were tested to identify some knock-out mutants that showed a loss of susceptibility or an increase in susceptibility to B. brassicae infestation in order to characterize the gene or genes responsible of the change in plant-insect interaction and look for homologous genes in other plant species, important from an economical point of view. For the investigation on plant’s targets interacting with M. euphorbiae effectors, it was decided to perform a yeast two hybrid assay between three potato aphid effectors, MeC002 and other two proteins that were showed from literature to increase aphid’s fecundity, and a library of pepper cDNA to identify possible aphid effectors-binding pepper proteins. The sequences of the three examined effectors were isolated from the potato aphid cDNA and cloned into the destination vectors containing the GAL4-Binding Domain to perform the yeast two hybrid assay. Yeast cells were transformed with two of the three generated baits and the different preys, representing the genes present in a normalized pepper cDNA library, and the screening for positive interactions was performed

Disease and Disorders of Freshwater Unionid Mussels: A Brief Overview of Recent Studies

The use of aquatic invertebrates in biomedical research and as environmental sentinels has dramatically grown in recent decades, with an increased need in understanding of comparative pathology. The Unionids freshwater mussels are a group of worldwide distributed bivalves residing small ditches and ponds, lakes, canals and rivers, often used as animal test in eco-toxicological studies. Once one of the most abundant bivalve molluscs in ancient rivers around the world, now many of them are declining in many countries and consequently are nearly extinct in many areas. The causes of this decline are not fully understood but alteration and degradation of the freshwater habitat seemed to play a central role. To date, link causality to the observed losses during episode of mussel die-offs has been more difficult to establish, and disease and pathogen presence have been scarcely considered. In this article we provide a brief overview of unionids freshwater mussel conservation status, also describing reported diseases and pathogens and illustrating a few relatively well-documented studies

The role of Need for Affect and Need for Cognition in self-evaluation

The primary aim of this study was to investigate whether individual differences in affective and cognitive orientation predict the relative importance of warmth-related and competence-related traits in self-evaluation. 99 participants (85 females) completed the Need for Affect and Need for Cognition scales. Later, participants rated the extent to which warmth- and competence-related traits described their own personality. In line with our hypotheses, affective people expressed more positive evaluations of warmth traits and more negative evaluations of cold traits relative to cognitive people, who expressed more positive evaluations of competence traits and more negative evaluations of incompetence traits. This differentiation has implications for self-evaluation processes and individual differences in affective and cognitive orientation

Serafino Zappacosta: An Enlightened Mentor and Educator.

With this article, the authors aim to honor the memory of Serafino Zappacosta, who had been their mentor during the early years of their career in science. The authors discuss how the combination of Serafino Zappacosta's extraordinary commitment to teaching and passion for science created a fostering educational environment that led to the creation of the "Ruggero Ceppellini Advanced School of Immunology." The review also illustrates how the research on the MHC and the inspirational scientific context in the Zappacosta's laboratory influenced the authors' early scientific interests, and subsequent professional work as immunologists

Gender influence on professional satisfaction and gender issue perception among young oncologists. A survey of the Young Oncologists Working Group of the Italian Association of Medical Oncology (AIOM)

Background: The professional gender gap is increasingly recognised in oncology. We explored gender issues perception and gender influence on professional satisfaction/gratification among young Italian oncologists. Methods: Italian oncologists aged 6440 years and members of the Italian Association of Medical Oncology were invited to participate in an online survey addressing workload/burnout, satisfaction in professional abilities and relations, relevant factors for professional gratification, and gender barriers. \u3c72 test for general association or \u3c72 test for trend was used to analyse the data. Results: 201 young oncologists participated in the survey: 67% female, 71% aged 30-40 years, 41% still in training and 82% without children. Women and men were equally poorly satisfied by the relations with people occupying superior hierarchical positions. There was heterogeneity between women and men in current (p=0.011) and expected future (p=0.007) satisfaction in professional abilities: women were more satisfied by current empathy and relations with colleagues and were more confident in their future managerial and team leader skills. The most important elements for professional gratification indicated by all participants were, in general, work-life balance (36%) and intellectual stimulation/research (32%); specifically for women, work-life balance (48%) and intellectual stimulation/research (20%); and specifically for men, career (29%) and social prestige/recognition (26%). Heterogeneity within the same gender emerged. For example, the elements indicated by men as the most important were intellectual stimulation/research (39%) and work-life balance (21%) in general, versus social prestige/recognition (24%) and career (24%), respectively, specifically for men (p<0.0001). More women versus men perceived gender issue as an actual problem (60% vs 38%, p=0.03); men underestimated gender barriers to women's career (p=0.011). Conclusions: Satisfaction in professional abilities varied by gender. Work-life balance is important for both women and men. Stereotypes about gender issues may be present. Gender issue is an actual problem for young oncologists, mostly perceived by women