Autor(es): Gouveia, Catarina
Data: 2024
Identificador Persistente: http://hdl.handle.net/10400.5/98696
Origem: Repositório da Universidade de Lisboa
Assunto(s): Vitis vinifera; Plasmopara viticola; Oomycete effectors; Subtilisin-like serine proteases; Serine protease inhibitors
Grapevine (Vitis vinifera L. subsp. sativa) is one of the most cultivated fruit crops worldwide. However, is highly susceptible to several pathogens which poses a severe threat to production yields. One of the most devastating diseases is the grapevine downy mildew caused by the obligate biotrophic oomycete Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni. This pathogen was introduced in Europe in the mid19th century and led to the near-collapse of European viticulture only some years later. To manage this disease several preventive applications of pesticides are used but more sustainable approaches such as breeding for resistance are being conducted. As such, most of the research in the field of grapevine-pathogen interaction is still focused on increasing knowledge of how the plant activates defence pathways. However, the success of the breeding programs depends also on a deep knowledge of this pathosystem. Not only on the identification of strong resistance-associated candidate genes for introgression from the resistance carriers and hybrids, but as well as on the interacting players in the plant-pathogen offense-defence, as plant defence proteins/ enzymes and pathogen effectors. The activation of the defence pathways and therefore the establishment of an incompatible interaction depends on the early recognition of pathogens. Among the groups of molecules, that play a role in pathogen recognition and signalling cascades, we focused on plant proteases, particularly in serine proteases, or the subtilisin-like proteases (SBT, subtilases) family. In grapevine, some subtilases were observed to have an enhanced gene expression upon infection with P. viticola in the tolerant cultivar “Regent” and susceptible cultivar “Trincadeira”, as VviSBT4.19. Moreover, some subtilases, as VviSBT5.3, were found to have their expression induced not only in response to the pathogen but also by jasmonic acid, a phytohormone involved in the signalling pathway in biotic stress response. Focusing on the pathogen, apoplastic effectors such as protease inhibitors are necessary for a successful invasion and counter defence. In this context, one hypothesis is that P. viticola may secrete protease inhibitors to block host serine proteases. This doctoral project’s main aim is to understand the role of subtilases in grapevine resistance and the role of serine-protease inhibitors secreted by P. viticola as pathogenicity effectors, particularly to understand the potential crosstalk between grapevine and P. viticola focusing on host subtilases and pathogen serine proteases inhibitors. Several objectives were defined for the analysis of host serine proteases as the analysis of VviSBT4.19 and VviSBT5.3 gene expression in V. vinifera genotypes with different tolerance and Rpv background in a time-course inoculation with P. viticola. Stable transformation of Solanum lycopersicum with constructs for overexpression of VviSBT4.19 and SlSBT4.14, a tomato homolog of VviSBT4.19 to access gene function, and characterization of these Solanum lycopersicum transformed lines. For the analysis of serine protease inhibitors, we identified P. viticola protease inhibitors from the serine family, performed gene expression analysis of these inhibitors and analysis of their subcellular localization and their effect on plant defence mechanism by agroinfiltration in N. benthamiana. Gene expression analysis of two candidate subtilases, VviSBT4.19 and VviSBT5.3a, was performed in seven cultivars with different levels of susceptibility to P. viticola (Chapter II). Two Vitis vinifera susceptible genotypes: “Chardonnay” and “MuellerThurgau” and five tolerant genotypes “Calardis blanc” (Rpv3–1, Rpv3–2), “Cabernet blanc” (Rpv3–1), “Regent” (Rpv3–1), “Solaris” (Rpv10, Rpv3-3) and “Sauvignac” (Rpv12, Rpv 3 − 1) were analysed. Moreover, two P. viticola isolates with different degrees of virulence were tested, avrRpv3 + which is an isolate unable to overcome Rpv3-based resistance and NW-10/16 which was isolated from ‘Regent’ in October 2016 in Neustadt/Weinstr. (Germany). Not only NW-10/16 can overcome the resistance observed in Rpv3 genotypes when infected with the avirulent isolate avrRpv3+, in the susceptible cultivars, “Chardonnay” NW-10/16 also exhibits an increase in sporulation. This higher sporulation on susceptible cultivars might indicate that the isolate NW-10/16 presents a higher aggressiveness. The subtilases display different patterns of expression with the VviSBT4.19 exhibiting only one peak of expression, while VviSBT5.3a presents a bimodal behaviour. Nevertheless, both subtilases presented a more pronounced response to the more aggressive P. viticola isolate, with the difference in gene expression being also dependent on the grapevine cultivar. Our results support the hypothesis that the two subtilases are involved in the early events of the defence mechanisms, being their transcription highly activated in response to a more aggressive P. viticola isolate. The P. viticola isolates were further characterized in Chapter III. The interaction of these isolates with 5 grapevine genotypes with different Rpv loci was analysed. In this chapter, P. viticola development, aggressiveness and the expression of serine protease inhibitors was assessed. The aim was to distinguish two P. viticola isolates through their infection strategy and growth stages, and possible determine if a different gene expression of serine protease inhibitors could be a cause of one of the causes of the increased aggressiveness. The results showed that the isolate NW-10/16 was able to overcome Rpv3 resistance but not Rpv12 resistance. Moreover, it can be considered more aggressive with increased sporulation and increased hyphae growth, with an acceleration in hyphae growth after 36 hours post inoculation. P. viticola serine protease inhibitors gene family was characterized with eight genes encoding for five proteins. From these five serine protease inhibitors, two present highly dynamic pattern of expression depending on the isolate and cultivar. The Pvit020s, a serine protease inhibitor gene that has three identical copies in the genome, plus one containing a mutation which alters 1 aminoacid, present a later peak of expression in the susceptible cultivar but earlier in tolerant cultivars. While Pvit026 has an both a higher expression level and an earlier peak of expression for NW-10/16 compared to avrRpv3+. When comparing the gene expression of both isolates, NW-10/16 presented a higher expression for Pvit026 in all cultivars and Pvit544 in the tolerant and resistant cultivars. The expression patterns indicate a role of serine proteases inhibitors in the pathogen’s counterattack against plant defence. Moreover, we can hypothesize that Pvit026 may be involved in increased aggressiveness, while Pvit544 might have a role as a facilitator to overcome resistance. The function of subtilisin-like serine protease VviSBT4.19 was studied in Chapter IV with the stable transformation of Solanum lycopersicum to obtain transgenic plant lines overexpressing the grapevine VviSBT4.19. To compare, the homolog subtilisinlike serine protease gene from tomato, SlSBT4.14 was also cloned and used for stable transformation. Cotyledons of S. lycopersicum “Hellfrucht” were co-incubated with A. tumefaciens AGL1 for stable transformation. No difference in calli morphology was observed between constructs (T-DNA containing VviSBT4.19, SlSBT4.14 or none). However, calli transformed with the transgene VviSBT4.19 presented a slightly faster regeneration. Three generations of transgenic plant lines were analyzed. For each construct two transgenic plant lines were studied. Morphologically, the overexpression of VviSBT4.19 altered the angle of the tomato leaves, which tended to grow with a vertical orientation. And plants overexpressing SlSBT4.14 tended to be shorter. Another major difference was found in flowering and fruit development. Plants overexpressing VviSBT4.19 and SlSBT4.14 tended to flower later with some not producing fruits and seeds. The resistance to Phytophthora infestans was evaluated for the transgenic plant lines and plants overexpressing SlSBT4.14 were found to be more susceptible. And plants overexpressing VviSBT4.19 tend to present a lower pathogen sporulation, and therefore tend to be less susceptible to P. infestans infection. These results indicate that both subtilases have possible functions in biotic stress response as well as plant development and reproduction. Moreover, the overexpression of VviSBT4.19 and SlSBT4.14 resulted in very different phenotypes regarding growth and biotic stress response, further indicating that these subtilases may participate in different mechanisms. Though the exact mechanism of action remains uncertain. As it was possible to determine in which plant processes these subtilases have probable functions, new experiments can be design and carried out more specifically, and thus further elucidating the subtilases roles in these processes. This thesis is divided in four chapters. The first chapter was written as a review article and the 3 following chapters were written as scientific articles and each has its own abstract, introduction, materials and methods, results and discussion, conclusion, acknowledgments and references.
