Subtilisin-like proteases and lipid signalling events play an important role in grapevine resistance to downy mildew: a systems biology approach

Uma abordagem de biologia de sistemas revela a importância de proteases serínicas e sinalização por lípidos na resistência da videira ao míldio

Andreia Figueiredo^1,*, Joana Figueiredo^1,2,3, Marisa Maia^1,2,3, Ana Rita Cavaco¹, Gonçalo Laureano¹, Rui Nascimento¹, Clemente da Silva¹, Ana Rita Matos¹ & Marta Sousa Silva^2,3

¹ Biosystems & Integrative Sciences Institute (BioISI), Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisboa, Portugal

² Laboratório de FTICR e Espectrometria de Massa Estrutural, Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisboa, Portugal  

³ Centro de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisboa, Portugal

(*E-mail: aafigueiredo@fc.ul.pt)

ABSTRACT

The reduction of pesticide use to control pests and diseases is one of modern agriculture’s demands. In the grapevine-Plasmopara viticola pathosystem, repeated preventive applications of fungicides occur during the vegetative period. The identification of specific mechanisms or molecules with bioactive fungicide properties may lead to new disease control strategies. Following an omics-based system biology approach we were able to identify both subtilisin-like proteases and lipid associated signalling events as key players on grapevine resistance mechanisms. Efforts are being conducted to characterize their role aiming at the definition of alternative control measures towards a sustainable viticulture.

RESUMO

Uma das prioridades para a sustentabilidade da agricultura é a redução da aplicação de pesticidas para o controlo de pragas e doenças. Atualmente é utilizada a aplicação preventiva repetida de fungicidas durante a época de cultivo para controlo do míldio da videira. A identificação dos mecanismos de resistência ou de moléculas com atividade biofungicida é de extrema importância para a definição de novas estratégias de controlo da doença. Utilizando uma abordagem de biologia de sistemas baseada em “Ómicas” (transcritómica, proteómica e metabolómica) foi possível identificar proteases de tipo subtilisina e eventos de sinalização associados a lipídios como mecanismos chave na resposta de resistência da videira ao míldio (Plasmopara viticola). Uma caracterização mais aprofundada destes mecanismos é essencial para a definição de medidas de controlo de doenças em prol de uma viticultura sustentável.

Palavras-chave: videira, míldio, subtilases, ácido jasmónico, resposta imunitária

ASSESSING GRAPEVINE-PLASMOPARA VITICOLA INTERACTION BY A SYSTEMS BIOLOGY APPROACH

Grapevine (Vitis vinifera L.) is the most widely cultivated and economically important fruit crop in the world, with deep ties to human culture for more than 5000 years. Today the wine industry plays a key role in many countries’ economy, with a global market size of over 29 billion euro (OIV, 2017). In 2016, Portugal reached the 11^th position in word wine production with 700 M€ revenue (OIV, 2017). Most of grapevine cultivars used for winegrowing are often affected by fungal and fungal-like pathogens causing severe harvest losses (Gessler et al., 2011). One of the most threatening diseases is the downy mildew caused by the obligate biotrophic oomycete Plasmopara viticola (Berk. et Curt.) Berl. et de Toni. This pathogen was introduced in Europe in the 19^th century becoming one of the most devastating diseases for today’s viticulture. European viticulture employs a large amount of agrochemicals to control fungal diseases and viticulture practices account for 65% of all fungicides used in agriculture (Eurostat report 2007, http://ec.europa.eu/eurostat). In the absence of treatments and favorable conditions, P. viticola may lead to losses up to 75% in one season (Buonassisi et al., 2017). Even though nearly all V. vinifera cultivars are susceptible to P. viticola, American grapevine species exhibit natural host resistance against this pathogen and have been used in breeding programs for resistance to grapevine pathogens resulting on V. vinifera varieties such as ‘Regent’ and ‘Solaris’ (Vitis International Variety Catalogue 2011; http://www.vivc.de). However, the defenses of these bred hybrids are being overcome by new P. viticola resistance-breaking isolates in Europe (Peressotti et al., 2010). Thus, for a sustainable viticulture it is imperative to get a deeper knowledge on the grapevine resistance mechanisms against P. viticola aiming at defining new friendly approaches to control the disease and to identify resistance candidates for increase of resistance durability after trait introgression in breeding programs.

By combining several ‘omic’ studies (transcriptome, proteome and metabolome) in a system biology approach (Figure 1) our group has contributed to a deeper knowledge on the early host responses to P. viticola attack. Using as a study model two grapevine cultivars, ‘Regent’ a resistant hybrid and ‘Trincadeira’, a Portuguese traditional variety highly susceptible to P. viticola, we have identified mechanisms and players that may play key roles on the establishment of the incompatible interaction.  

The resistant cultivar ‘Regent’ presents higher accumulation of proteins and metabolites related to primary and secondary metabolism and biosynthetic machinery that may pay for the fitness cost of a constitutive resistance (Figueiredo et al., 2008, 2017). Moreover several genes associated with plant immunity are constitutively expressed, as the case of those encoding subtilisin-like proteases.

In the resistant genotype, as soon as 6 hours post inoculation (hpi) a broad reprogramming of the transcriptome occurs with major changes associated to the stress response and signalling events, such as subtilisin-like proteins, IQ calmodulin-binding region- Apoptosis regulator Bcl-2 protein, cyclophilin, leucine-rich repeat (LRR) receptor like proteins, PR10 proteins and CBL-interacting serine/threonine (Figueiredo et al., 2012; Monteiro et al., 2013). Also, both the precursor of jasmonates in the octadecanoid cascade linolenic acid (C18:3) as well as several transcripts and proteins related to lipid signalling events are more accumulated in ‘Regent’ (Ali et al., 2012; Figueiredo et al., 2012, 2015, 2017; Guerreiro et al., 2016). At the metabolome level the response of the resistant genotype is also characterized by accumulation of phenolic metabolites such as trans-caftaric acid (caffeoyl derivative), trans-fertaric acid (feruloyl derivative) and quercetin-3-O-glucoside (Ali et al., 2012).

In summary, our results point out to two key mechanisms differentiating ‘Regent’ and ‘Trincadeira’, one is related to the involvement of serine proteases of the subtilisin family in pathogen recognition and immunity activation and the other is associated to lipid signalling events, particularly to the involvement of jasmonic acid (JA) in grapevine resistance against a biotrophic pathogen as P. viticola (Figure 1).

The signalling pathways associated to grapevine and P. viticola interaction are still poorly understood. In plant defense against pathogens, phytohormones such as jasmonates and salicylic acid (SA) have received considerable attention (reviewed in Berens et al., 2017). It is generally assumed that SA is involved in the activation of defense responses against biotrophic and hemi-biotrophic pathogens, as well as the establishment of systemic acquired resistance, whereas inducible defense against leaf-chewing insects and necrotrophic microbes is mediated by jasmonic acid (JA)-dependent signalling (reviewed in Glazebrook, 2005; Antico et al., 2012). These generalities are disputed in grapevine, as JA signalling has been implicated in resistance against biotrophs, such as powdery and downy mildew (Hamiduzzaman et al., 2005; Belhadj et al., 2006; Trouvelot et al., 2008; Antico et al., 2012; Figueiredo et al., 2015; Guerreiro et al., 2016).

The importance of lipids in signalling processes, during the interaction between grapevine and P. viticola, has been highlighted in our recent studies (Ali et al., 2012; Figueiredo et al., 2015, 2017; Guerreiro et al., 2016). We have shown that Vitis vinifera ‘Regent’ presents an early up-regulation of enzymes involved in JA biosynthesis and a later up-regulation for JA signalling enzymes (Guerreiro et al., 2016), together with a higher accumulation of both JA, JA-ILE and SA in the first hours of interaction (Guerreiro et al., 2016). These results provide strong evidences that JA may participate, in resistant cultivar, as a signalling molecule in defense response against P. viticola acting synergistically with SA. We have also shown both a higher increase in H₂O₂ accumulation and lipid peroxidation in the resistant genotype (Figueiredo et al., 2017). Non-specific lipid transfer proteins as well as plastid lipid associated proteins (also named fibrillins) were also accumulated in ‘Regent’ suggesting lipid transport. Fribrilins were shown to act as supports for building lipid droplets that contain free fatty acids, carotenoids, phytols, quinones, and other lipophilic compounds and some of them could be damaged due photooxidative conditions. In stressed plants, an association between fibrillins levels and JA production was observed. The storage of JA precursors, as C18:3, would allow a rapid JA production after oxidative stress. Our results suggest that at the first hours after grapevine inoculation with P. viticola lipid associated signalling events occur in the resistant genotype, and that both JA and SA participate in defense response against this biotrophic pathogen. A study by Polesani et al. (2010) also corroborates our results on this pathosystem by showing an increase of jasmonates and their biosynthetic enzymes in the resistant grapevine specie V. riparia after inoculation with P. viticola.

GRAPEVINE SUBTILISIN-LIKE PROTEASES AND P. VITICOLA RESISTANCE

Subtilisin-like proteins or subtilases are gaining particular attention in the context of plant-pathogen interactions. Subtilases are serine proteases that are synthesized as inactive proteins, suffer glycosylation during protein maturation and are secreted to the plant extracellular matrix (ECM) where they accumulate and presumably recognize and process substrates (Bykova et al., 2006; Chichkova et al., 2004; Cedzich et al., 2009, Figueiredo et al., 2017). Plant ECM is where the first host-pathogen interaction and recognition events take place. Thus accumulation of subtilases in the ECM rise the possibility of an important involvement during pathogenesis. In grapevine, the first clues highlighting subtilase participation in defence mechanisms were reported by Figueiredo and co-workers (2008), when comparing resistant (‘Regent’) and susceptible (‘Trincadeira’) genotypes prior and post-inoculation with Plasmopara viticola. A subtilisin-like protease (XM_010660203.1), identified as a cucumisin was constitutively expressed in the resistant genotype and increased its expression after P. viticola inoculation (Figueiredo et al., 2008, 2012; Monteiro et al., 2013). Also in this pathosystem, Gindro et al. (2012) have shown that by treating plants with serine protease inhibitors they became more sensitive to P. viticola. Recent studies revealed that grapevine subtilases are encoded by a multigenic family comprising 87 genes that encode 109 proteins (Figueiredo et al., 2016, 2017a). We have analysed the expression modulation of 14 grapevine subtilases during P. viticola inoculation (Figueiredo et al., 2016). The selected grapevine subtilases shared sequence similarity with Arabidopsis SBT3.3 and tomato P69, already described as participating in plant immunity events (reviewed in Figueiredo et al., 2017b) and were located near  P. viticola resistance associated loci (Rpv). It is clear that in the susceptible genotype there is a delay in the induction of all the subtilase genes studied. In the resistant genotype subtilases, in particular the subtilase VvSBT4.19 (XM_010660203.1), are highly expressed at 6hpi, suggesting an involvement in the activation of defence responses.

THE LINK BETWEEN SUBTILASE AND PHYTOHORMONE SIGNALLING IN GRAPEVINE RESPONSE TO P. VITICOLA

The first clue into a link between subtilases and phytohormones signaling (SA and JA pathways) was shown by the study of Salzman et al. (2005) where a subtilase increased its expression after Sorghum bicolor elicitation with methyl jasmonate (MeJA), SA, and aminocyclopropane carboxylic (ACC). It was observed a SA- and MeJA-mediated induction of putative homologs of genes composing the entire octadecanoid pathway for JA synthesis (Salzman et al., 2005).

Moreover, despite little being known about plant subtilases’ substrates, the substrates known are  a prosystemin (Schaller and Ryan, 1994) and the leucine-rich repeat protein (LRP) (Tornero et al., 1996). Systemin is generated after prosystemin cleavage by subtilases and plays a fundamental role in response to herbivore damage or mechanical wounding through the systemic signalling pathway activation of plant defence genes. Systemin appears to be involved in the activation of the octadecanoid pathway for jasmonic acid biosynthesis, particularly by the activation of a lipase in receptor cell membranes resulting in the release of linolenic acid, the production of jasmonic acid, and the activation of proteinase inhibitor genes (Bergey et al., 1996). Moreover, it was later shown that systemin is perceived at the cell surface by a leucine-rich repeat receptor-like kinase that induces the JA synthesis pathway at the site of wounding as a prerequisite for systemic defence gene induction (Howe and Schaller, 2008; Ryan, 2000; Wasternack et al., 2006).

These results open new insights into the involvement of subtilases in the control of signalling pathways associated to lipids. Further work must be conducted to validate this hypothesis, and particularly in the grapevine- P. viticola pathosystem, were both subtilases and JA seem to be key players in the establishment of an incompatible interaction.

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Acknowlegdments

This work was supported by projects PEst-OE/BIA/UI4046/2014, PEst-OE/QUI/UI0612/2013, UID/MULTI/00612/2013, SFRH/BD/116900/2016, PD/BD/131030/2017, Investigator FCT program IF/00819/2015 (Fundação para a Ciência e a Tecnologia, FCT/MCTES/PIDDAC, Portugal) and Joana Figueiredo PhD grant from Universidade de Lisboa.

Received/recebido: 2017.12.15

Accepted/aceite: 2018.04.09