Dissertação apresentada para obtenção do grau de Doutor em Engenharia Civil na Faculdade de Engenharia da Universidade do Porto, sob a orientação do Prof. Doutor Adão da Fonseca

"R. solanacearum" is a devastating bacterial pathogen that infects "Solanaceae" spp. such as tomato, eggplant or banana. A functional T3SS is required for virulence and more than 70 putative effectors have been described, although only few have been studied. This thesis focuses on a five-member gene family of effectors named "awr". We demonstrated that awr gene family is extremely conserved among R. solanacearum strains but also present in other plant pathogens such as Acidovorax or Burkholderia spp. and even present in the human pathogen B. pseudomallei. Virulence of a Ralstonia mutant strain devoid of all awr genes was tested on tomato, eggplant and Aradidopsis. Plant growth of quintuple mutant strain was considerably reduced in natural hosts, indicating a role in virulence, but remained unchanged in Arabidopsis. Col-0 infection with Pseudomonas syringae DC3000 heterologously expressing each AWR was also performed. While presence of some AWRs in Pseudomonas did not have an effect on plant growth, others (like AWR5) dramatically reduced the pathogen multiplication, pointing out a possible plant detection. In order to unravel the functions of AWR proteins, they were transiently expressed by means of Agrobacterium in non-host Nicotiana spp. Upon AWR expression, necroses took place to different extents on the plant leaves. AWR5 induced the strongest necrosis, resembling an HR phenotype which was later confirmed by TB/DAB staining and by RT-PCR of specific HR marker genes. Furthermore, a strong reduction in yeast cells was experimented upon several AWR protein expressions which indicate that the mechanisms that might be altered by these effector proteins is conserved among eukaryotes and hence reinforces their role in virulence. AWR4 appeared not to be toxic in this model organism and for that reason we sought to decipher some of the plant targets of this AWR protein as a start point. Out of more than 60 interacting clones were sequenced after a yeast-two hybrid screening with Arabidopsis root cDNA from R. solanacearum challenged plants. Among them, several defense-related proteins were found: phenylalanine ammonia-lyase, MPK6, DMR6 or KIN10. In order to find other key genes for AWR activity, the AWRs that displayed a strong yeast toxicity were heterologously produced in both E. coli and R. solanacearum to be ready to be employed as a bait for plant protein complexes that will be analysed by mass spectrometry. In summary, AWR are highly conserved effectors that play an important role in both pathogenesis and plant recognition as they reduce P. syringae virulence and trigger an HR-like phenotype in non-host plants. Deciphering effector function will open promising avenues towards the design of new strategies to control R. solanacearum. / R. solanacearum és un patogen bacterià capaç d’infectar diferents solanàcies com ara la tomaquera, la patatera, l’alberginiera o el plataner. Aquest fitopatogen injecta més de 70 proteïnes efectores en la cèl•lula vegetal hoste, tot i que només algunes han sigut ja estudiades. Aquesta tesi es centra en una família multigènica d’efectors: els AWRs. Els estudis científics duts a terme durant aquesta tesi van demostrar que la família de AWR no només estava altament conservada en el llinatge de R. solanacearum sinó que també es trobava present en altres fitopatògens o inclús en el patogen humà Burkholderia pseudomallei. A més a més, diferents assajos de patogenicitat en tomaquera i alberginiera van provar que els gens awr presentaven un paper clar en virulència per aquests hostes. Contràriament, la presència d’aquestes proteïnes en la planta model Arabidopsis thaliana produïen una disminució en la capacitat infectiva/multiplicativa. Això indicaria una dualitat dels efectors AWR depenent del context que ens trobem, ja sigui contribuint a la patogenicitat del bacteri o bé éssent reconeguts per la planta i així disminuint la patogenicitat bacteriana. Per tal de desentranyar les funcions de les proteïnes AWR, es van expressar de forma transitòria a la planta model no-hoste Nicotiana spp. L’expressió d’algunes proteïnes AWR va provocar una forta necrosi de les fulles que s’assemblaria a una resposta hipersensible. Mitjançant diferents tincions i assajos de PCR en temps real es va corroborar que l’AWR5 presentava aquest tipus de mort cel•lular programada. L’elevada toxicitat d’algunes AWRs es va demostrar també en llevat. En el transcurs d’aquesta tesi també s’ha realitzat un crivellatge en doble híbrid per tal de buscar proteïnes dianes de la planta per a l’AWR4 (la menys tòxica). A més a més, es va posar a punt l’expressió dels AWRs a E. coli o bé a R. solanacearum per tal d’abordar altres tècniques que permetin una millor cerca d’interactors en el futur.