Light perception influences disease resistance in plants

- Essay in plant science -

Light is a key environmental factor in plant development. Light-regulated processes in plants range from seed germination to photoperiodic flowering. In addition, light also contributes to plant defense against pathogens and is necessary for the expression of various defensive genes (Karpinski et al. 2003; Bechtold et al. 2005; Roberts and Paul 2006).

The Salicylic Acid (SA) pathway is essential in the orchestration of the plant defense signaling. The role of light in the control of SA-dependent hypersensitive response as well as in the expression of the Pathogenesis Related 1 (PR-1) gene has been studied (Genoud et al. 2002; Zeier et al. 2004). In addition, Arabidopsisplants grown under low light or dark conditions are compromised in their local as well as systemic resistance to Pseudomonas syringae (Genoud et al. 2002; Zeier et al. 2004). Genetic evidence supporting the role of phytocrhome-mediated light perception and signaling in plant defense against P.syringae came from studies with phytochrome A (phyA) and phytochrome B (phyB) mutants. Mutations in phyA or phyB affected the hypersensitive response (HR) and blocked the SA-induced PR-1 expression (Genoud et al. 2002).

Casein kinase 2 (CK2) is a serine/threonine kinase with ubiquitous functions ranging from cell cycle regulation to circadian rhythms (Mizoguchi et al. 2006; Salinas et al. 2006). In plants, CK2 is involved in light-regulated gene expression and photoperiod sensitivity (Lee et al. 1999; Takahashi et al. 1998; Mizoguchi et al. 2006). The nuclear activity of CK2 was shown to increase in response to SA accumulation (Hidalgo et al. 2001).

These reports highlight the importance of light in the outcome of plant-pathogen interactions. However, the molecular mechanisms that link light signaling with disease resistance in plants remains unknown. Here, I hypothesize that CK2 may play a role in the light-dependent response of Arabidopsis to Pseudomonas syringae infection. This hypothesis is based on the following observations. First, a cis-element called the as-1 is required for the transcriptional activation of SA-responsive genes and the binding of transcription factors to the element is mediated by CK2 (Hidalgo et al. 2001). Second, CK2 is involved in circadian rhythms and the photoperiod response of Arabidopsis and rice (Sugano et al. 1998; Takahashi et al. 1998; Sugano et al. 1999; Mizoguchi et al 2006). Third, the protein abundance of a CK2 regulatory subunit, CKB4, is circadian regulated (Perales et al. 2006). This suggests that CK2 activity may vary depending on the time of the day and the season.

In this short review, I discuss about the possible effects that light signaling and clock mutations may have in the CK2-mediated transcription of SA-responsive genes upon infection with P. syringae. In addition, a possible role for CKB4 in circadian modulation of CK2 activity and consequently in the SA-mediated defense response to pathogen infection is described.

Pseudomonas syringae is a plant pathogen that infects many species causing local necrotic lesions in infected tissues.

P. syringae is a rod shaped, Gram-negative bacterium that is represented by 50 different pathovars. Some pathovars of P. syringae infect crops of highly agronomic importance including wheat and barley (P. atrofaciencs), soybean (P. glycinea), sunflower (P. elianthi), Brassica (P. maculicola), tomato (P. tomato) (Agrios G.N., Plant Pathology, Elsevier Academic Press, 2005). P. syringae as well as many other Gram-negative bacteria have developed the ability to deliver virulence factors into the host cells through a conserved type three secretion system (TTSS) (Alfano and Collmer 2004). Some of these virulence factors from P. syringae pv. tomato (Pst) strain DC3000 were found to be suppressors of the SA-mediated basal defense (DebRoy et al. 2004).

The SA-mediated signaling pathway is essential for plant defense

Plant infection by pathogens triggers a wide range of defenses in order to avoid colonization. In the infected leaf, these include the development of necrotic lesions and expression of defensive genes with antimicrobial activities such as the pathogenesis related proteins (PR genes). In uninfected tissue, PR genes are subsequently activated. This response is associated with a long-lasting resistance known as the systemic acquired resistance (SAR) (Reviewed by Durang and Dong 2004). SA plays a critical role in the establishment of SAR. There are two groups of genes that are activated in response to SA accumulation: rapid induced genes including glutathione s-transferases (GSTs) and late responsive genes such as the PR genes.

The activity of CK2 is important for the SA-mediated induction of defense gene expression.

SA increased the CK2 activity in tobacco plants (Hidalgo et al. 2001). A CK2 inhibitor, 5,6-Dichloro-1-(beta-D-ribofuranosyl) benzimidazole (DRB), prevented the binding of transcription factors to the as1-like sequence in response to SA (Hidalgo et al. 2001). The TGA2 transcription factor was identified as a target protein for CK2 activity in the SA-mediated defense pathway. The TGA transcription factors are necessary for the establishment of SAR in response to SA accumulation (Kang and Klessig 2005). These results indicate that CK2 plays key roles in the control of the SA-mediated expression of defense genes.

The participation of CK2 in the regulation of gene transcription in plants

A cis-element called the G-box is required for induction of genes controlled by light (Giuliano et al. 1988; Sibéril et al. 2001). CK2 phosphorylated a family of transcription factors, G-box binding factors (GBFs), in vitro and affected binding activity of the GBFs to the G-box sequences (Klimczack et al. 1995). Further evidence suggesting a role of CK2 in light-regulated gene expression was demonstrated by anti-sense suppression of CK2 alpha subunit gene expression in Arabidopsis (Lee et al. 1999). The phosphorylation of the bZIP transcription factor Opaque2 (involved in maize seed development) by CK2 affected Opaque2 DNA binding properties (Ciceri et al. 1997). Two single-MYB transcription factors, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) play key roles in photoperiodic flowering, responses to light and circadian clock functions in Arabidopsis (Sugano et al. 1998 and Sugano et al. 1999). CK2 phosphorylates both LHY and CCA1 proteins in vitro. Overexpression of CKB3 shortens the period length under continuous light and accelerates flowering under short day (Daniel et al. 2004). These results suggest a central role of CK2 function in the control of circadian rhythms (Sugano et al. 1999; Daniel et al. 2004; Mizoguchi et al. 2006).

Light influences the outcome of SA-mediated defense of Arabidopsisagainst P.syringae infection.

Light modulates many aspects of plant development and is also important for plant defense against pathogens, being required for the activation of many defense genes and the modulation of the hypersensitive response (HR) (Asai et al. 2000; Chandra-Shekara et al. 2006). Several reports have demonstrated that PR-1 gene expression induced by SA and accumulation of SA in response to bacterial pathogen are dependent on light (Genoud et al. 2002; Zeier et al. 2004). In addition, Arabidopsis plants grown under low light or dark conditions are compromised in their local as well as systemic resistance to P. syringae (Genoud et al. 2002; Zeier et al. 2004). The requirement of light for the SA-mediated induction of defense gene expression and establishment of HR in response to P.syringae can be attributed via effects from both photosynthesis related processes (mainly reactive oxygen species (ROS) production under high light) and phytochrome-mediated light perception and signal transduction pathways. Genetic evidence supporting the role of phytochrome-mediated light perception and signaling in plant defense against P.syringae came from studies with phytochrome A (phyA) and phytochrome B (phyB) mutants. Mutations in phyAor phyB affected the hypersensitive response (HR) and blocked the SA-induced PR-1 expression (Genoud et al. 2002). A stronger effect of phyA phyB double mutant on the SA-mediated defense pathway suggests that light has a quantitative effect on SA pathway and plant defense. The authors also demonstrated that although light regulated the SA pathway, the opposite did not occur (Genoud et al. 2002).

In another report, the interaction of Arabidopsis with an avirulent strain of P. syringae pv. maculicola was also found to be light-dependent (Zeier et al. 2004). Infection of Arabidopsis with P. syringae pv. maculicola in the dark resulted in increased apoplastic bacterial growth and thus reduced local resistance as compared to that during light conditions (Zeier et al. 2004). Furthermore, accumulation of jasmonic acid and production of the phytoalexin camalexin (an antimicrobial compound) in response to P. syringae pv. maculicola was enhanced in the dark. Light also influenced the onset of systemic acquired resistance (SAR). SAR progression in response to P. syringae pv. maculicola was lost in the absence of light (Zeier et al. 2004).

It has been shown that a mutation in a gene, CONSTITUTIVE SHADE-AVOIDANCE 1 (CSA1), mediating the shade avoidance response also affected disease susceptibility (Faigon-Soverna et al. 2006). The CSA1 gene encodes a Toll/Interleukin 1 receptor with nucleotide binding site and leucine-reach-repeats (TIR-NBS-LRR). The csa1 mutant is impaired in red light signaling and enhanced the apoplastic growth of P.syringae. It is proposed that CSA1 alters PHYB signaling through an interaction with the TIR-NBS-LRR gene RPS4 (Faigón-Soverna et al. 2006).

CK2 is involved in clock function.

In many prokaryotic and eukaryotic organisms, biological clocks mediate the response of several physiological and molecular processes to diurnal changes in environmental conditions. Circadian rhythms persist with a period close to 24 hours in the absence of any environmental time cue and are generated by an endogenous timing mechanism. The basic principles of circadian clocks have been addressed for many organisms such as cyanobacteria, Neurospora, Arabidopsis, mice and humans. The clock consists in oscillating molecules that form a negative auto-regulatory feedback loop (Barak et al. 2000; Harmer et al. 2001; Salomé and McClung 2004; Más 2005). Phosphorylation of these molecules is necessary for normal circadian function in humans, Drosophila and Neurospora where CK2 is the main protein kinase (Barak et al. 2000; Harmer et al. 2001; Salomé and McClung 2004; Más 2005; Mizoguchi et al. 2006). In Arabidopsis, the oscillatory molecules that compose the clock include: the two single-MYB transcription factors LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), as well as the PSEUDO RESPONSE REGULATOR (PRR) containing protein PRR1 (also known as TOC1) (Alabadí et al. 2001; Alabadí et al. 2002; Mizoguchi et al. 2002).

CK2 phosphorylates CCA1 and over-expression of CKB3 (a beta subunit of CK2) abolished circadian rhythms (Daniel at al. 2004). Although CK2 has an essential role in circadian function, the mRNA abundance of its regulatory subunits does not appear to be under circadian control. This raises the question about how CK2 modulates its activity at different times of the day. One possible mechanism could be at the protein level, in which the protein abundance of CK2 regulatory subunits is circadian regulated. According to this, the involvement of the clock in the circadian regulation of the protein abundance of a CK2 regulatory subunit, CKB4, has been reported recently (Perales and Más 2006). Over-expression of CKB4increased CK2 activity and caused period-shortening and phase shift in the expression of clock-controlled genes peaking at different times of the day (Perales and Más 2006; Portolés and Más 2007).

Furthermore, CKB4-ox plants displayed short hypocotyls and flowered earlier than wild type under SDs. Also, the expression of the core clock components TOC1 and CCA1 was affected, indicating that CKB4 act very close to the central oscillator (Portolés and Más 2007).

The components of the oscillator receive environmental inputs to allow synchronization of the clock with the external environment (McClung et al. 2002). Three classes of photoreceptors provide with light input to the oscillator affecting the period and/or the phase of the rhythms: the phytochromes (PHYA-E), cryptochromes (CRY1 and 2) and a class of blue light photoreceptors with a Light-Oxygen and Voltage (LOV) domain called ZITUPLE (ZTL) and LOV, KELCH Protein 2 (LKP2) (Devlin and Kay 2001).

PHYA and PHYB have a role in red light input to the clock, whereas PHYA is involved in blue light input together with CRY1 (Somers et al. 1998). The analysis of circadian photo-perception in double and triple phytochrome and cryptochrome mutants have shown that PHYA, PHYB, PHYD and PHYE additively convey red light input to the clock, whereas CRY1 and CRY2 have a redundant role in blue light input (Devlin and Kay 2000).

Mutations in the gene ZTL resulted in long period circadian phenotype that was dependent on light intensity (Somers et al. 2000) and its over-expression resulted in arrhythmicity (Somers et al. 2004). A role for LKP2 in the circadian clock of Arabidopsis has been demonstrated. When over-expressed, LKP2 produced arrythmic phenotypes in several clock outputs, long hypocotyls under both blue and red light and late flowering (Schultz et al. 2001).

Addressing the role of CKB4 in SA-mediated defense

CK2 protein is a tetramer complex constituted by two alpha catalytic subunits and two beta regulatory subunits. CK2 subunits mainly display nuclear and cytosolic localization although they have been found in the mitochondria and chloroplast as well (Salinas et al. 2006). In the Arabidopsis genome, four genes coding for alpha subunits and four genes coding for beta subunits have been identified, being all of them expressed (Salinas et al. 2006). The CK2 beta regulatory subunit 3 (CKB3) directly interacts with CCA1 and promotes its phosphorylation (Sugano et al. 1998; Sugano et al. 1999; Daniel et al. 2004). Recently, the role of CKB4 in clock function has been investigated. Over-expression of CKB4 increased CK2 activity and shortened the free running period of clock-controlled genes. Surprisingly, CKB4 itself is under circadian control displaying rhythms in its protein abundance (Perales and Más 2006).

Considering the importance that CK2 has on the SA-mediated induction of defense gene transcription, what is the role of CKB4 in SA-mediated defense of Arabidopsis against P.syringae infection? Addressing the role that CKB4 may have on CK2 activity in SA-mediated defense will imply a possible and direct connection between circadian rhythms and plant-pathogen interactions.

Perspectives

Mutations in light signaling and clock components of Arabidopsismay affect the outcome of P.syringae infection.

Taking together the above considerations, it is clear that light perception through phytochromes has an essential role in both SA-mediated defense against pathogen infection and also circadian clock function. Recently, the isolation of a gene that is not only induced in response to P. syringae infection but also circadian regulated has been reported (Sauerbrunn et al. 2004). The gene was named PCC1 for Pathogen and Circadian Controlled 1. The rhythmically expression of PCC1 was altered in plants over-expressing the clock component CCA1 (Sauerbrunn et al. 2004). This work revealed for the first time the existence of a cross talk between plant-defense signaling and circadian rhythms. However, the mechanism involved in this crosstalk remains unknown. The conceptual model suggested here places CK2 as a possible and feasible link between light-mediated SA accumulation and circadian function. This model presents an exciting framework to look at plant-pathogen interactions from a new perspective in which the light environment has an essential role in the outcome of plant disease-resistance. Although not addressed here, the production of Reactive Oxygen Species (ROS) in plants is highly dependent on light. ROS or free radicals are produced by electron transfer reactions and are also used by the cell in antibacterial and antifungal defense. In photoautotrophic organisms, the chloroplast is the main source of ROS. Plants thus protect themselves of ROS damage by the generation of daily/circadian rhythms in the activity of antioxidants enzymes as well as in the production of low molecular weight antioxidants. What is the effect of circadian improper function in clock mutants for oxidative stress management? Circadian rhythms of protective antioxidant enzymes have been described in phylogenetically distant organisms implying an essential role of circadian systems to fight against externally induced and internal generated ROS (Hardeland et al. 2003).

Based on these considerations, a full set of light signaling and clock mutants await to be tested in the susceptibility or resistance to pathogen infection. Furthermore, the presence of a complete circadian system in the fungi Neurospora crassa is leading to the identification of homologous clock components in many pathogenic fungi such as Gibberella zeae (Fusarium) and Magnaporthe grisea. It is possible to predict also that mutations in clock components of pathogenic fungi will impact in the infection process under different light conditions.

Only recently, the molecular mechanisms of light-sensing and plant defense against pathogen infection started to be explored. Stomatal aperture and closure is regulated by several environmental factors such as light intensity, air humidity and CO2 concentration, internal rhythms in stomata aperture and closure is imposed by the circadian clock. Both internal and environmental factors converge in stomatal regulation to maximize photosynthesis efficiency and to prevent water loss. Stomata have important roles in the plant innate immune system by restricitng bacterial invasion (Reviewd by Underwood et al. 2007).

Microarray analysis have shown that phytocrhome activity influence the expression of many plant disease resistance genes (Tepperman et al. 2001; Devlin et al. 2003). As reported by Genoud et al. in 2002, the growth of an incompatible strain of P. syringae was enhanced in the phyA phyB double mutant. In addition, the Arabidopsis mutant phytochrome signaling 2 (psi2), was reported to develop light-dependent necrotic lesions similar to those observed during the hypersensitive response caused by pathogen in incompatible interactions (Genoud et al. 1998). Very recently, the growth of an incompatible strain of P. syringae was increased in the csa1 mutant, which it is affected in the shade-avoidance response (Faigon-Soverna et al. 2006).

The above information strongly suggests that photomorphogenic processes and defense responses are interconnected.

Figure 1. A model for CK2 at the interface between plant defense and circadian clock function. Pathogen infection triggers SA accumulation leading to an induction in the expression of defense genes such as PR-1 and PCC1. At the same time, SA accumulation increases CK2 activity. CK2 phosphorylation is required for the binding of transcription factors to the as-1 element of genes involved in the SA pathway. CK2 also phosphorylates the clock component CCA1, thus altering circadian rhythms in the expression of many genes such as PCC1. An interesting aspect of the system is that the protein abundance of CK2 regulatory subunits is circadian regulated (Perales et al. 2006). Light and its signaling through phytochromes have essential roles in the modulation of SA accumulation in response to pathogen infection as well as in circadian-clock function.

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