Confección de la herramienta

Senescence is a developmentally induced process, controlled by the maturity of the leaf, but in addition, it is a stress induced process that can be triggered by external factors. One of these external factors that induces senescence is the infection by pathogenic species. Upon infection by a pathogen, a plant will initiate a range of responses, that include, but are not exclusive to, senescence. Since these two processes are linked, the detection and response to pathogens is interwoven with the regulatory elements that control senescence processes. Infection by the necrotrophic

fungusBotrytis cinerea induces senescence in the area immediately surrounding the

lesion site in Arabidopsis (Swartzberg et al., 2008). As such, a brief description of

the infection process and response toBotrytis cinerea is appropriate.

1.4.1. Botrytis cinerea is a classical necrotrophic plant pathogen

A wide variety of pathogens infect Arabidopsis, each with different lifestyles and methods of infection. All pathogens exist with the aim of sequestering nutrients from Arabidopsis, but use different methodologies to extract them. The most well known example of this is the contrast between necrotrophs and biotrophs (Oliver & Ipcho, 2004). Necrotrophs kill cells through toxins or induced cell-death and then feed on the nutrients released, while biotrophs feed on living tissue and therefore aim to maintain cell viability to harvest nutrients (Panstruga, 2003; Glazebrook, 2005). Within these two broad categories are far more divisions, with different pathogens using different strategies to feed off plant tissue.

Botrytis is a fungal necrotroph with a broad host range (van Kan, 2006). Often referred to as grey mould, due to the formation of grey condia on infected tissue,

it is a major crop pathogen causing losses in nearly 200 species (Williamson et al.,

2007). As a necrotroph, its primary mode of action is to consume nutrients released from dead plant cells killed using toxins and lytic enzymes. As such, the infectious cycle of Botrytis is relatively straightforward compared to many other pathogens (Schumacher & Tudzynski, 2012).

Conidia germinate on plant tissue before forming appresorial-like structures which

penetrate the thick plant cuticle (Williamsonet al., 1995; van Kan, 2006). At this

point the Botrytis establishes a primary infection site by killing cells in the imme- diate vicinity. The infection can then enter a latent phase, however this is normally

reserved for flowers or developing fruit (Holz et al., 2007). More commonly, after

a short lag phase Botrytis infection will lead to a wet lesion and begin to spread. Botrytis excretes a mix of endopolygalacturonases for degradation of plant cell wall

pectin and proteases for degradation of plant proteins (Have et al., 1998; Wubben

et al., 1999; Karset al., 2005; Espinoet al., 2010), the which provide the primary nu-

trients for Botrytis growth. Alongside this Botrytis secretes oxalates to acidify the environment to generate the optimum environment for the pathogen degradation

enzymes (Manteauet al., 2003; van Kan, 2006).

The degradation of plant cell walls and proteins generates a spreading lesion from the initial infection site which generates the necrotic lesion of a Botrytis infection

(Zhang et al., 2014). As such, these enzymes are required for full pathogenicity

and establishment of the infection (Have et al., 1998; Kars et al., 2005). Toxins

such as botrydial are secreted into the vicinity to kill cells before they can mount

an appropriate response (Reino et al., 2004; Fernández-Acero et al., 2007). The

concentration of toxins directly correlates with virulence, suggesting they are critical for infection.

Botrytis is often regarded as one of the only ‘true necrotrophs’, that is it infects a broad host range by a relatively simple mechanism of cell degradation followed by consumption of the nutrients released. As such, it does not subvert the immune system using effector proteins as many biotrophic pathogenic organisms will. Having said this, Botrytis does hijack host machinery for establishment of the full infection. In fact, full pathogenicity of Botrytis requires host participation (van Kan, 2006;

Williamsonet al., 2007).

Botrytis is known to induce the hypersensitive response in Arabidopsis using an

unknown elicitor (Govrin & Levine, 2000; Govrin et al., 2006). The hypersensitive

response is a form of programmed cell death triggered in response to pathogen attack to inhibit the spread of biotrophic organisms by preventing access to living cells (Jones & Dangl, 2006). However, Botrytis releases elicitors in the area surrounding

the hyphae to promote cell death and therefore lesion size (Govrinet al., 2006).

Similarly, Botrytis produces reactive oxygen species (ROS) at hyphal tips and

plasma membrane during infection (Schoutenet al., 2002). Histochemical staining

oxide and autophagosome-like vesicles at the host-pathogen interface (van Baarlen

et al., 2007). Furthermore, the concentration of ROS at the infection site is corre-

lated with pathogenicity (Tiedemann, 1997), suggesting the production of ROS is a positive mechanism for infection by Botrytis. Rapid transient oxidative stress at an infection site is one of the key elements of pathogen response by Arabidopsis (Apel & Hirt, 2004), so it seems odd that Botrytis contributes to the production of ROS. However, Botrytis appears to be immune to the high concentrations of hydrogen per- oxide (Temme & Tudzynski, 2009) possibly due to Botrytis producing catalases for

clearance of hydrogen peroxide (Schouten et al., 2002). This suggests that Botrytis

has co-opted the production of ROS for infection establishment.

Finally, Botrytis can induce premature senescence. Expression of the senescence

specific geneSAG12 increases in leaves infected with Botrytis, suggesting senescence

is promoted around the lesion (Swartzberget al., 2008). Expression ofIPT from the

SAG12 or SAG13 promoter in tomato causes a severe delay in senescence, but also

confers enhanced resistance to Botrytis infection (Swartzberget al., 2006), suggesting

senescence is critical for establishment of a full infection. As such, it is believed Botrytis exploits the host senescence mechanism to facilitate its own infection.

1.4.2. Recognition of Botrytis cinerea by Arabidopsis thaliana

Plants have a suite of surface receptors embedded in their extracellular membrane which identify microbial proteins and markers (known as Microbe Associated Molec- ular Markers, or MAMPS; Boller & Felix, 2009). These receptors are often referred to as plasma membrane (PM)-resident pattern recognition receptors, or PRRs. PRRs contain an extracellular domain which is capable of binding to a particular protein or oligosaccharide unique to microbes. Evolution has driven these to be molecules that are critical for the microbe but not present in plants, such as flagellin or chitin. In addition, a number of these receptors recognise molecules released upon damage to the plant architecture, such as pectin released from plant cell wall degradation, known as Damage Associated Molecular Marker (DAMP).

Upon treatment with the MAMPs or DAMPs, PRRs are activated and the intracel- lular domain will begin to trigger the response, known as PAMP-triggered immunity (PTI). Many of these surface proteins identified have been Receptor-Like Kinases (RLKs), with an intracellular Ser/Thr kinase domain (Shiu & Bleecker, 2003), which indicates they may have an intracellular enzymatic activity in addition to their ex- tracellular recognition activity. Perhaps the most well known example of this is the flg22 receptor FLAGELLIN-SENSITIVE 2 (FLS2; Gómez-Gómez & Boller, 2000), whose intracellular domain rapidly forms a complex with BRI1-ASSOCIATED RE-

CEPTOR KINASE1 (BAK1) after recognition of the flg22 (Chinchilla et al., 2007;

Sunet al., 2013). In turn, the FLS2/BAK1 complex binds to BOTRYTIS INDUCED

responses such as a MAPK signalling cascade, rapid increase in Ca2+, production

of phytoalexins and deposition of callose to reinforce the cell wall and close stomata

(Anilet al., 2013).

As a fungus, Botrytis does not utilise flagellin and does not contain flg22. As such, identification of MAMPs for Botrytis has taken some time. The fungal protein chitin is a necessary component of fungal cell walls and therefore largely immutable in evo- lutionary terms, indicating it was likely to be a MAMP for Botrytis. Eventually the protein CHITIN ELICITOR BINDING PROTEIN (CEBiP) was identified in rice, which was shown to recognise chitin and trigger immune responses such as MAPK activation, reactive oxygen species generation and expression of defence related genes

(Miyaet al., 2007).

1.4.3. Transcriptional changes during Botrytis cinerea infection

Upon detection of a pathogen at the exterior, plant cells will initiate a range of responses to combat the new threat. Defence mechanisms come in many forms, but all require the synthesis of new proteins and therefore pathogenic response is an active process rather then a passive resistance. The activation of defence pathways

can be observed by whole genome expression changes (Windramet al., 2012).

Initially, there is a lag phase where very little change in gene expression is observed for approximately 12 hours post infection. This is shortly followed by rapid increase in transcription of genes relating to biosynthesis of ethylene synthesis, particularly

1-AMINOCYCLOPROPANE-1-CARBOXYLATE (ACC) SYNTHASES(ACS2 and

ACS6), which catalyse the rate limiting step in ethylene biosynthesis. Ethylene is

one of the critical hormones involved in the response to necrotrophic pathogens, with mutations of ethylene signalling cause enhanced susceptibility to necrotrophs

(Thommaet al., 1999). Therefore expression of genes relating to ethylene synthesis

helps generate ethylene that is used to trigger the ET-mediated pathogen responses

(Thommaet al., 1999; Díazet al., 2002).

Shortly after this, expression of genes that relate to ethylene response signalling

dramatically increase (Windramet al., 2012). This is concurrent with the upregula-

tion of a number of genes relating to jasmonic acid signalling, which acts in concert with ethylene. The JA/ET signalling pathway promotes a significant proportion

of the responses to necrotrophic pathogens such as Botrytis cinerea, therefore this

represents the beginning of the active response to the infection.

At around 14 hours post infection, expression of genes relating to photosynthetic mechanisms such as chlorophyll biosynthesis reduce, alongside an increase in the expression of genes relating to chlorophyll metabolism and degradation. The down- regulation of photosynthetic mechanisms has been observed in a number of plant- pathogen interactions. It is suggested to be a mechanism by which the plant reallo-

2010).

Later, changes in expression of a number of metabolism related genes occur, pre- sumably to clear metabolites that accumulate during Botrytis infection, such as

camalexin, flavonoids and glucosinolates (Denby et al., 2004; Kliebenstein & Rowe,

2008). Metabolites such as these are critical to establishing a full defence toBotrytis

cinerea, therefore rapid synthesis of a sufficient number of enzymes for production

of this are critical.

During infection, Arabidopsis reinforces the cell wall to physically restrict the

spread of Botrytis (Ellis & Turner, 2001; Ellis et al., 2002). In response to the

infection, Arabidopsis induces expression of the cellulase synthase genesCeSA1 and

CeSA3, which presumably reinforce the cell wall for enhanced resistance to Botrytis.

A mutation of CeSAV3, known as cev1, shows constitutive activation of the the

jasmonic acid and ethylene pathways, thus enhancing the expression of a number of

stress responsive genes that confer enhanced resistance toBotrytis cinerea infection

(Ellis & Turner, 2001; Ellis et al., 2002). This may indicate cell wall modifications

share a direct link to phytohormone signalling, although the mechanism behind this is unclear.

Botrytis cinerea produces a number of toxins whose role is to lyse the cell in order

for the contents to be released (Amselem et al., 2011). In response, Arabidopsis

produces a number of genes that may relate to the catabolism of these toxins, such as glutathione s-transferases (GST), two of which have been implicated in detoxification

of synthetic toxins (Dixon et al., 2009).

Finally, there are a number of classical biotic stress response marker genes that are frequently expressed in response to necrotrophic pathogens. Subsequently these

genes have often been used as a marker of pathogen response. These includePLANT

DEFENSIN GENE 1.2 (PDF1.2), which is rapidly expressed in response to activa-

tion of the ethylene and jasmonic acid signalling pathways (Penninckxet al., 1998).

PDF1.2 and other PDF genes appear are similar to other eukaryotic defensin genes

and have anti-fungal properties (Broekaertet al., 1995; Stotzet al., 2009).