The pathogenicity of Plasmopara viticola: a review of evolutionary dynamics, infection strategies and effector molecules.

Oomycetes are filamentous organisms that resemble fungi in terms of morphology and life cycle, primarily due to convergent evolution. The success of pathogenic oomycetes lies in their ability to adapt and overcome host resistance, occasionally transitioning to new hosts. During plant infection, these organisms secrete effector proteins and other compounds during plant infection, as a molecular arsenal that contributes to their pathogenic success. Genomic sequencing, transcriptomic analysis, and proteomic studies have revealed highly diverse effector repertoires among different oomycete pathogens, highlighting their adaptability and evolution potential.The obligate biotrophic oomycete Plasmopara viticola affects grapevine plants (Vitis vinifera L.) causing the downy mildew disease, with significant economic impact. This disease is devastating in Europe, leading to substantial production losses. Even though Plasmopara viticola is a well-known pathogen, to date there are scarce reviews summarising pathogenicity, virulence, the genetics and molecular mechanisms of interaction with grapevine.This review aims to explore the current knowledge of the infection strategy, lifecycle, effector molecules, and pathogenicity of Plasmopara viticola. The recent sequencing of the Plasmopara viticola genome has provided new insights into understanding the infection strategies employed by this pathogen. Additionally, we will highlight the contributions of omics technologies in unravelling the ongoing evolution of this oomycete, including the first in-plant proteome analysis of the pathogen.

Deep into the Apoplast: Grapevine and Plasmopara viticola Proteomes Reveal the Secret Beneath Host and Pathogen Communication at 6 h After Contact.

The apoplast is the first hub of plant-pathogen communication where pathogen effectors are recognized by plant defensive proteins and cell receptors, thus activating signal transduction pathways. As a result of this first contact, the host triggers a defense response that involves the modulation of extra- and intracellular proteins. In grapevine-pathogen interactions, little is known about the trafficking between extra- and intracellular spaces. Grapevine is an economically important crop that relies on heavy fungicide use to control several diseases, and a deeper knowledge on the activation of its immune response is crucial to define new control strategies. In this study, we focused on the first 6 h postinoculation with Plasmopara viticola to evaluate grapevine proteome modulation in the apoplast. The in planta P. viticola proteome was also assessed to enable a deeper understanding of plant-pathogen communication. Our results showed that several plant mechanisms are triggered in the tolerant grapevine cultivar Regent after inoculation, such as oomycete recognition, plant cell wall modifications, reactive oxygen species signaling, and secretion of proteins to disrupt oomycete structures. On the other hand, P. viticola proteins related to development and virulence were the most predominant. This pioneer study highlights the early dynamics of cellular communication in grapevine defense that leads to the successful establishment of an incompatible interaction.

An in-planta comparative study of Plasmopara viticola proteome reveals different infection strategies towards susceptible and Rpv3-mediated resistance hosts.

Plasmopara viticola, an obligate biotrophic oomycete, is the causal agent of one of the most harmful grapevine diseases, downy mildew. Within this pathosystem, much information is gathered on the host, as characterization of pathogenicity and infection strategy of a biotrophic pathogen is quite challenging. Molecular insights into P. viticola development and pathogenicity are just beginning to be uncovered, mainly by transcriptomic studies. Plasmopara viticola proteome and secretome were only predicted based on transcriptome data. In this study, we have identified the in-planta proteome of P. viticola during infection of a susceptible ('Trincadeira') and a Rpv3-mediated resistance ('Regent') grapevine cultivar. Four hundred and twenty P. viticola proteins were identified on a label-free mass spectrometry-based approach of the apoplastic fluid of grapevine leaves. Overall, our study suggests that, in the compatible interaction, P. viticola manipulates salicylic-acid pathway and isoprenoid biosynthesis to enhance plant colonization. Furthermore, during the incompatible interaction, development-associated proteins increased while oxidoreductases protect P. viticola from ROS-associated plant defence mechanism. Up to our knowledge this is the first in-planta proteome characterization of this biotrophic pathogen, thus this study will open new insights into our understanding of this pathogen colonization strategy of both susceptible and Rpv3-mediated resistance grapevine genotypes.

Transcriptomic and methylation analysis of susceptible and tolerant grapevine genotypes following Plasmopara viticola infection.

Downy mildew, caused by the biotrophic oomycete Plasmopara viticola, is one of the most economically significant grapevine diseases worldwide. Current strategies to cope with this threat rely on the massive use of chemical compounds during each cultivation season. The economic costs and negative environmental impact associated with these applications increased the urge to search for sustainable strategies of disease control. Improved knowledge of plant mechanisms to counteract pathogen infection may allow the development of alternative strategies for plant protection. Epigenetic regulation, in particular DNA methylation, is emerging as a key factor in the context of plant-pathogen interactions associated with the expression modulation of defence genes. To improve our understanding of the genetic and epigenetic mechanisms underpinning grapevine response to P. viticola, we studied the modulation of both 5-mC methylation and gene expression at 6 and 24 h post-infection (hpi). Leaves of two table grape genotypes (Vitis vinifera), selected by breeding activities for their contrasting level of susceptibility to the pathogen, were analysed. Following pathogen infection, we found variations in the 5-mC methylation level and the gene expression profile. The results indicate a genotype-specific response to pathogen infection. The tolerant genotype (N23/018) at 6 hpi exhibits a lower methylation level compared to the susceptible one (N20/020), and it shows an early modulation (at 6 hpi) of defence and epigenetic-related genes during P. viticola infection. These data suggest that the timing of response is an important mechanism to efficiently counteract the pathogen attack.

Uncovering the role of DNA methyltransferases in grapevine - Plasmopara viticola interaction: From genome-wide characterization to global methylation patterns.

Epigenetic regulation has recently gained prominence in the field of plant-pathogen interactions, providing a deeper understanding of the molecular mechanisms associated with plant infection. In grapevine interaction with pathogens, epigenetic regulation still remains a black box. In this work, we characterized grapevine DNA methyltransferase gene family and identified nine DNA methyltransferases genes across eight grapevine chromosomes coding for 17 proteins. We also assessed the modulation of global cytosine methylation and gene expression levels of these genes with the aim of establishing a connection between DNA methylation and grapevine resistance towards downy mildew. Our results revealed that, in the incompatible interaction, an early hypomethylation, coupled with downregulation of DNMT and CMT genes occurs very early after pathogen inoculation. Additionally, the compatible interaction is characterized by a hypermethylation at 6hpi. A temporal delay is evident between the shifts in DNA methyltransferases gene expression in both compatible and incompatible interactions which in turn may be reflected in the global methylation percentage. Overall, we present the first evidence of an epigenetic regulation role in grapevine defense against P. viticola.

The grapevine aspartic protease gene family: characterization and expression modulation in response to Plasmopara viticola.

MAIN CONCLUSION: Grapevine aspartic proteases gene family is characterized and five VviAPs appear to be involved in grapevine defense against downy mildew. Grapevine (Vitis vinifera L.) is one of the most important crops worldwide. However, it is highly susceptible to the downy mildew disease caused by Plasmopara viticola (Berk. & Curt.) Berl. & De Toni. To minimize the use of fungicides used to control P. viticola, it is essential to gain a deeper comprehension on this pathosystem and proteases have gained particular interest in the past decade. Proteases were shown to actively participate in plant-pathogen interactions, not only in the processes that lead to plant cell death, stress responses and protein processing/degradation but also as components of the recognition and signalling pathways. The aim of this study was to identify and characterize the aspartic proteases (APs) involvement in grapevine defense against P. viticola. A genome-wide search and bioinformatics characterization of the V. vinifera AP gene family was conducted and a total of 81 APs proteins, coded by 65 genes, were found. VviAPs proteins can be divided into three categories, similar to those previously described for other plants. Twelve APs coding genes were selected, and expression analysis was conducted at several time-points after inoculation in both compatible and incompatible interactions. Five grapevine APs may be involved in grapevine tolerance against P. viticola. Our findings provide an overall understanding of the VviAPs gene family and establish better groundwork to further describe the roles of VviAPs in defense against P. viticola.

Two sides of the same story in grapevine-pathogen interactions.

Proteases are an integral part of plant defence systems, and their role in plant-pathogen interactions is unequivocal. Emerging evidence suggests that different protease families contribute to the establishment not only of hypersensitive response, priming, and signalling, but also of recognition events through complex proteolytic cascades. Moreover, they play a crucial role in pathogen/microbe-associated molecular pattern (PAMP/MAMP)-triggered immunity as well as in effector-triggered immunity. However, despite important advances in our understanding of the role of proteases in plant defence, the contribution of proteases to pathogen defence in grapevine remains poorly understood. In this review, we summarize current knowledge of the main grapevine pathosystems and explore the role of serine, cysteine, and aspartic proteases from both the host and pathogen point of views.

Defense and Offense Strategies: The Role of Aspartic Proteases in Plant-Pathogen Interactions.

Plant aspartic proteases (APs; E.C.3.4.23) are a group of proteolytic enzymes widely distributed among different species characterized by the conserved sequence Asp-Gly-Thr at the active site. With a broad spectrum of biological roles, plant APs are suggested to undergo functional specialization and to be crucial in developmental processes, such as in both biotic and abiotic stress responses. Over the last decade, an increasing number of publications highlighted the APs' involvement in plant defense responses against a diversity of stresses. In contrast, few studies regarding pathogen-secreted APs and AP inhibitors have been published so far. In this review, we provide a comprehensive picture of aspartic proteases from plant and pathogenic origins, focusing on their relevance and participation in defense and offense strategies in plant-pathogen interactions.

Synthesis and biological effects of small molecule enhancers for improved recombinant protein production in plant cell cultures.

Histone deacetylases are involved in chromatin remodelling and thus play a vital role in the epigenetic regulation of gene expression. HDAC inhibitors alter the acetylation status of histone and non-histone proteins to regulate various cellular events such as transcription. Novel HDAC inhibitors were designed and synthesised to promote higher levels of recombinant protein production in tobacco cell cultures. The effect of these chemical enhancers on the epigenetic profiles in plant cells has been evaluated by molecular docking, in vitro and in vivo studies. The addition of these novel enhancers led to an increase in histone H3 acetylation levels that promoted an increase in the accumulation levels of the recombinant protein in cell culture. These results can pave the way for the application of these enhancers to improve the production of high value products in plant cell based systems.

Low Protease Content in Medicago truncatula Cell Cultures Facilitates Recombinant Protein Production.

Medicago truncatula is an established model for studying legume biology. More recently, it has also been exploited as a Molecular Farming platform for the production of recombinant proteins, with the successful expression of fungal and human proteins in plants and cell suspension cultures of this species. One of the challenges that now must be overcome is the degradation of final products during production and downstream processing stages. In the M. truncatula genome, there are more than 400 putative protease-encoding genes, but to date, the proteolytic content of Medicago cell cultures has not been studied. In this report, the proteolytic activities that can potentially hamper the successful production of recombinant proteins in this system are evaluated. The potential proteases responsible for the degradation of target proteins are identified. Interestingly, the number of proteases found in Medicago spent medium is considerably lower than that of the well-established tobacco bright yellow 2 (BY-2) system. Papain-like cysteine proteases are found to be the major contributors to recombinant protein degradation in Medicago. This knowledge is used to engineer a cell line with reduced endogenous protease activity by expressing a selective protease inhibitor, further improving this expression platform.

Addition of a histone deacetylase inhibitor increases recombinant protein expression in Medicago truncatula cell cultures.

Plant cell cultures are an attractive platform for the production of recombinant proteins. A major drawback, hindering the establishment of plant cell suspensions as an industrial platform, is the low product yield obtained thus far. Histone acetylation is associated with increased transcription levels, therefore it is expected that the use of histone deacetylase inhibitors would result in an increase in mRNA and protein levels. Here, this hypothesis was tested by adding a histone deacetylase inhibitor, suberanilohydroxamic acid (SAHA), to a cell line of the model legume Medicago truncatula expressing a recombinant human protein. Histone deacetylase inhibition by SAHA and histone acetylation levels were studied, and the effect of SAHA on gene expression and recombinant protein levels was assessed by digital PCR. SAHA addition effectively inhibited histone deacetylase activity resulting in increased histone acetylation. Higher levels of transgene expression and accumulation of the associated protein were observed. This is the first report describing histone deacetylase inhibitors as inducers of recombinant protein expression in plant cell suspensions as well as the use of digital PCR in these biological systems. This study paves the way for employing epigenetic strategies to improve the final yields of recombinant proteins produced by plant cell cultures.

Putting the Spotlight Back on Plant Suspension Cultures.

Plant cell suspension cultures have several advantages that make them suitable for the production of recombinant proteins. They can be cultivated under aseptic conditions using classical fermentation technology, they are easy to scale-up for manufacturing, and the regulatory requirements are similar to those established for well-characterized production systems based on microbial and mammalian cells. It is therefore no surprise that taliglucerase alfa (Elelyso®)-the first licensed recombinant pharmaceutical protein derived from plants-is produced in plant cell suspension cultures. But despite this breakthrough, plant cells are still largely neglected compared to transgenic plants and the more recent plant-based transient expression systems. Here, we revisit plant cell suspension cultures and highlight recent developments in the field that show how the rise of plant cells parallels that of Chinese hamster ovary cells, currently the most widespread and successful manufacturing platform for biologics. These developments include medium optimization, process engineering, statistical experimental designs, scale-up/scale-down models, and process analytical technologies. Significant yield increases for diverse target proteins will encourage a gold rush to adopt plant cells as a platform technology, and the first indications of this breakthrough are already on the horizon.