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How Plasmopara viticola breaks through the grapevines’ defense mechanisms

Grapevine downy mildew is evoked by the oomycete Plasmopara viticola and causes major problems in vineyards all over the world. To achieve infection P. viticola has to overcome the defense mechanisms of the plant. A real evolutionary battle is going on between P.viticola and the grapevine. In recent years, their complex interplay is slowly unraveled, but there is still a long way to go.

The grapevine, like all plants, has developed over the course of evolution a complex defense mechanism against microorganisms. This innate immune system detects potential intruders. In a reaction to damage or environmental stresses, the plant starts to produce hormones such as jasmonic acid, salicylic acid and ethylene that set in motion a variety of defense reactions such as the production of reactive oxygen species (ROS), closing of the stomata, and phytoalexins or deposition of callose to strengthen the cell wall. Microorganisms such as the downy mildew pathogen P. viticola on the other hand have also improved their attacking mechanisms during evolution, creating an evolutionary arms race between pathogens and plants.

Hypha and haustoria in plant cells

Downy mildew hypha with haustoria (spheres in the cells) growing in the leaf of Arabidopsis thaliana, observed by bright-field microscopy.
Adapted from Emmanuel Boutet via CC BY-SA 2.5

Parasite

Oomycetes like P. viticola need water to germinate and are therefore often referred to as ‘water molds’. During rain, zoospores of P. viticola are splashed on the leafs of the grapevine and swim to the stomata. Subsequently, the germ tube of the zoospore enters the stoma and forms a hypha inside the leaf. The hypha is a long branching structure that extends further into the leaf of the grapevine and forms haustoria in the cells. These haustoria are structures that penetrate through the plants’ cell wall which enables P. viticola to siphon nutrients from the cell and grow even larger. American grapevine species are able to block this parasitic behavior by the programmed cell death of the affected cells. Vitis vinifera varieties are not able to do this and are therefore more vulnerable to P. viticola1,2.

Cross section leaf and infection by oomycete zoospore

Cross section of a grapevine leaf. Infection of grapevine leaf by P. viticola zoospore and the formation of a hypha and haustorium

Evolutionary arms race

When under attack by microorganisms, the first action of the grapevines’ innate immune system is to detect pathogen associated molecular patterns (PAMPs). These PAMPs are (part of) molecules that are typical for microbes and are not associated with the plants’ own cells. Examples are lipopolysaccharide for gram-negative bacteria, double-stranded RNA for viruses and β-glucans that are found in fungal cell walls. When these molecules are recognized by the plant, the innate immune system is triggered. This immune response is called the PAMP-triggered immunity (PTI) and is mediated by the above mentioned plant hormones.

During evolution pathogens like P. viticola have adapted to circumvent PTI by secreting ‘effectors’. Effectors are specialized proteins that increase the susceptibility of the plant to the pathogen. This process is called the ‘effector triggered susceptibility’ (ETS) and increases the virulence of the pathogen. However, as in all evolutionary processes, the plant also adapted to this, and has developed ways to recognize the effectors by using resistance proteins. Activation of these proteins leads to an immune response called effector triggered immunity (ETI). The defense reactions of the ETI are similar to those of the PTI, but are often more intense, resulting in cell dead at the site of infection. This programmed cell death is a defense mechanism of the plant to stop the growth of P. viticola, and is also called the hypersensitive response.

Pathogens and plants continuously improve and develop new mechanisms of ETS and ETI, resulting in an evolutionary arms race2-4.

Interaction between P. viticola and plant cell.

Interaction between the effectors produced by P. viticola and the immune system of the plant cell.

EFFECTORS

P. viticola isolates from Italy, France and China were characterized and were shown to produce different sets of effectors3. These are candidate effectors, which means that they are identified based on their conserved domain and are not functionally studied. New studies on the function and subcellular localization of subsets of P. viticola candidate effectors are already performed5,6, but there is still a long way to go. Most P. viticola effectors are classified based on their structural domains in protein families, but have not yet been functionally studied.

Nevertheless, there is a good general idea on how the different effector families contribute to the virulence of P. viticola. That is, the produced effectors can be transported into the plant cell where they weaken the plants’ immune defenses by interfering with the plant hormone pathways7. In addition, they can also exert their function on the outside of the plant cell by damaging the cell wall, cell membrane, interfering with receptors, or by preventing the actions of secreted defense proteins from the plant. The first group is called cytoplasmic effectors and the latter are called apoplastic effectors.

Downy mildew on grapevine leaf

Downy mildew on grapevine leaf.
Adapted from Rude via CC BY-SA 3.0

Cytoplasmic Effectors

There are three predominant families of cytoplasmic effector proteins identified in P.viticola, the RXLR effector proteins, the CRN effector proteins and the YxSLK effector proteins2. The proteins in these families are characterized by a conserved domain. For example, the RXLR proteins have a RXLR domain that consists of a conserved Arginine (R), random amino acid (X), Leucine (L) and Arginine (R) motif. These domains are required for entry of the effector protein into the plant cell, and target the effectors to a specific subcellular location such as the nucleus, the cytosol, the membrane, or even the chloroplasts and mitochondria in the cell5. Researchers use this conserved domain also to recognize new putative cytoplasmic effectors in the transcriptome of P. viticola2,3. Inside the cell, the rest of the protein subsequently exerts its effects to suppress defense responses such as the PTI, and promote virulence of the oomycete2-4. Of note, many resistance proteins have the ability to recognized RXLR motifs. Proteins of the family of RXLR effectors are therefore often important for the resistance of the plant to P. viticola. As such, many RXLR effectors are counterproductive, and hinder the infection and are therefore called avirulence factors8,9.

Apoplastic effectors

Apoplastic effectors do not enter the plant host cell, but interact with extracellular targets and receptors on the surface of the plant cell to facilitate the infection. The most prevalent apoplastic effectors are glycoside hydrolases, peptidases and protease inhibitors, but extracellular toxins like NLPs, and degradative enzymes such as cellulases, cutinases, pectate lysases and pectin esterases are also expressed by P. viticola3. The degradative enzymes target different components of the plants’ cell wall and membrane to enable degradation and penetration of the cell.

The variety of these effector proteins shows the complexity of the pathogen – host interaction. For example, the protease inhibitors that are expressed by P. viticola target the proteases that are secreted by the grapevine, that again were produced to degrade the effectors that were secreted by P. viticola.

An overview of all these (cytoplasmic and apoplastic) effectors is shown in Table 1.

Table 1. Effectors that are produced by Plasmopara viticola to aid in the infection of grapevine2-4,6  

Cytoplasmic effectorsFunction  
RXLR effectorsDisturbing and inhibiting the PTI and other plant defense responses in the plant cell
CRN effectorsDisturbing and inhibiting the PTI and other plant defense responses in the plant cell
YxSLK effectorsDisturbing and inhibiting the PTI and other plant defense responses in the plant cell
Apoplastic effectorsFunction  
CellulaseDegradation of cellulose in the cell wall
CutinaseDegradation of cutin, a polymer that protects the epidermis of the plant.
Elicitin-like proteinsA toxic protein that induces necrosis and the hypersensitive response. Sequesters sterols from the plant cell.
Trypsin-like proteinsProteases that cleave proteins. Involved in the response mechanism against plant defenses.
Glycoside hydrolasesBroad family of proteins that are involved in the hydrolysis (break down) of glycosidic bonds.
LipasesDegradation of lipids.
NLPsNLPs induce necrosis and the ethylene response in plant cells. May also be involved in zoospore attachment and suppressing PAMP recognition.
PectinesterasesBreakdown of the plant cell wall
Pectate lyasesCleaves pectic acid, a degradation product of pectin.
ProteasesBreakdown of proteins (also called ‘peptidases’)
Protease inhibitorsBlock the breakdown of proteins by inhibiting proteases

Identification of resistance genes

The large differences in susceptibility in Vitis species is due to the fact that P. viticola is native to North America. In the 1870s, P. viticola was introduced in Europe and resulted in massive damage to the European vineyards. The European Vitis vinifera cultivars are very susceptible, in contrast to the North American Vitis species that due to co-evolution acquired a natural resistance. American Vitis species therefore express a set of resistance proteins that are not produced in the Vitis vinifera varieties. There is large interest in the grapevine genes that encode these resistance proteins, for they provide the needed resistance to P. viticola. Alternatively, genes that are expressed in susceptible, but not in resistant grapevine species might confer susceptibility to P. viticola. To discover these genes, the interaction between P. viticola and susceptible and resistant Vitis species are studied and their transcriptomes are compared.

Toffolatti and colleagues compared the transcriptome of the susceptible Pinot noir cultivar to that of the resistant hybrid Bianca and the resistant Vitis vinifera cultivar Mgaloblishvili that shows unique resistance traits. They found a susceptibility gene, and a large list of novel candidate genes that might confer resistance to P. viticola2. However, the function of most of these transcripts is unknown, and functional tests will have to be performed to determine if and how these transcripts contribute to the resistance to P. viticola.

Breeding of new resistant varieties

Knowledge about the exact immune response mechanisms would benefit the (molecular) breeding of new resistant grapevine varieties. New varieties can be selected based on their ability to express resistance proteins, which would shorten the selection process.

READ ALSO: Breeding, genetic modification and CRISPR in the vineyard

Alternatively, new developed cultivars can be subjected to a set of effectors that are known to trigger the ETI in resistant cultivars. For example, Brilli and colleagues showed that the RXLR effector named ‘PVITv1008311’ triggers the ETI in Vitis riparia. This resulted in necrotic spots on the leafs and reduced growth of P.viticola. This same effector did not trigger an immune response in the susceptible Vitis vinifera cultivar Pinot noir. This shows that, although the exact immune response triggered by this effector in Vitis riparia is still unknown, it can be used to screen for new resistant grape varieties10.

Future research

The interplay between P. viticola and the grapevine is complex, and consists of multiple layers of virulence and defense mechanisms. Future research will probably show that this evolutionary battle is even more complex than we already know, but will also give breeders the right tools to design and produce new resistant varieties. In addition, when the immune mechanisms are known, it may also be possible to design alternatives to copper and chemical sprays for effectively controlling grapevine downy mildew.

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References
1. Toffolatti SL, De Lorenzis G, Costa A, Maddalena G, Passera A, et al. Unique resistance traits against downy mildew from the center of origin of grapevine (Vitis vinifera). Sci Rep. 2018 Aug 21;8(1):12523. https://doi.org/10.1038/s41598-018-30413-w.
2. Toffolatti SL, De Lorenzis G, Brilli M, Moser M, Shariati V, et al. Novel Aspects on The Interaction Between Grapevine and Plasmopara viticola: Dual-RNA-Seq Analysis Highlights Gene Expression Dynamics in The Pathogen and The Plant During The Battle For Infection. Genes (Basel). 2020 Feb 28;11(3). pii: E261. doi: https://doi.org/10.3390/genes11030261.
3. Yin L, Li X, Xiang J, Qu J, Zhang Y, et al. Characterization of the secretome of Plasmopara viticola by de novo transcriptome analysis. Physiological and Molecular Plant Pathology. 2015 July;91:1-10. https://doi.org/10.1016/j.pmpp.2015.05.002
4. Jiang RHY and Tyler BM. Mechanisms and Evolution of Virulence in Oomycetes. Annu. Rev. Phytopathol. 2012 50:295-318. https://doi.org/10.1146/annurev-phyto-081211-172912
5. Liu Y, Lan X, Song S, Yin L, Dry IB, Qu J, Xiang J, Lu J. In Planta Functional Analysis and Subcellular Localization of the Oomycete Pathogen Plasmopara viticola Candidate RXLR Effector Repertoire. Front Plant Sci. 2018 Apr 13;9:286. https://doi.org/10.3389/fpls.2018.00286. eCollection 2018.
6. Schumacher S, Grosser K, Voegele RT, Kassemeyer HH, Fuchs R. Identification and Characterization of Nep1-Like Proteins From the Grapevine Downy Mildew Pathogen Plasmopara viticola. Front Plant Sci. 2020 Feb 13;11:65. https://doi.org/10.3389/fpls.2020.00065. eCollection 2020.
7. Han X, Kahmann R. Manipulation of Phytohormone Pathways by Effectors of Filamentous Plant Pathogens. Front Plant Sci. 2019 Jun 26;10:822. https://doi.org/10.3389/fpls.2019.00822. eCollection 2019.
8. Mestre P, Carrere S, Gouzy J, Piron M-C, Tourvieille de Labrouhe D, et al. Comparative analysis of expressed CRN and RXLR effectors from two Plasmopara species causing grapevine and sunflower downy mildew. Plant Pathology. 2015 Oct 11;65(5):767-781. https://doi.org/10.1111/ppa.12469
9. Amaro TM, Thilliez GJ, Motion GB, Huitema E. A Perspective on CRN Proteins in the Genomics Age: Evolution, Classification, Delivery and Function Revisited. Front Plant Sci. 2017 Feb 3;8:99. https://doi.org/10.3389/fpls.2017.00099. eCollection 2017.
10. Brilli M, Asquini E, Moser M, Bianchedi PL, Perazzolli M, Si-Ammour A. A multi-omics study of the grapevine-downy mildew (Plasmopara viticola) pathosystem unveils a complex protein coding- and noncoding-based arms race during infection. Sci Rep. 2018 Jan 15;8(1):757. https://doi.org/10.1038/s41598-018-19158-8.

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