PSKR1 and PSY1R-mediated regulation of plant defense responses

Plant peptide signaling is an upcoming topic in many areas of plant research. Our recent findings show that the tyrosine sulfated peptide receptors PSKR1 and PSY1R are not only involved in growth and development but also in plant defense. They modulate salicylate- and jasmonate-dependent defense pathways in an antagonistic manner and this phenomenon might be dependent on the age and developmental stage of the plant. Here we discuss how the endogenous peptides might integrate growth, wounding, senescence and the opposing defense pathways against biotrophic and necrotrophic pathogens for increased fitness of the plant.     

A role for PSK signaling in wounding and microbial interactions in Arabidopsis

PSK‐α is a disulfated peptide that acts as a growth factor in plants. PSK‐α is derived from preproproteins which are encoded by five PSK precursor genes in Arabidopsis thaliana (L.) Heynh and is perceived by leucine‐rich repeat receptor kinases. Arabidopsis has two PSK receptor genes, PSKR1 and PSKR2. Although ligand and receptor are well characterized, the biological functions of PSK signaling are not well understood. Using reporter lines and receptor knockout mutants of Arabidopsis, a role for PSK signaling in biotic interactions and in wounding was analyzed. Treatment of Arabidopsis leaves with the fungal elicitor E‐Fol, or the fungal pathogens Alternaria brassicicola and Sclerotinia sclerotiorum resulted in induction of PSK2 and PSKR1 as shown by promoter:GUS analysis. Wounding of hypocotyls or leaves induced PSK3:GUS, PSK5:GUS and PSKR1:GUS expression indicating that PSK precursor genes are differentially regulated in response to specific stresses. The receptor knockout lines pskr1‐3 and pskr2‐1 showed significantly reduced photosynthesis in response to the fungal elicitor E‐Fol which indicates that fungal defence is impaired. pskr1‐3 plants further showed reduced growth of crown galls after infection with Agrobacterium tumefaciens. A role for PSK signaling in Agrobacterium tumefaciens tumor growth was supported by the finding that PSK precursor genes and PSKR1 are expressed in crown galls. Overall, the results indicate that PSK signaling may play a previously undescribed role in pathogen or herbivore interactions and is crucial for Agrobacterium‐induced cell proliferation in crown gall formation.     

The peptide growth factor, phytosulfokine, attenuates pattern-triggered immunity

Pattern-triggered immunity (PTI) is triggered by recognition of elicitors called microbe-associated molecular patterns (MAMPs). Although immune responses may provide good protection of plants from pathogen attack, excessive immune responses have negative impacts on plant growth and development. Thus, a good balance between positive and negative effects on the immune signaling network is important for plant fitness. However, little information is known about the molecular mechanisms that are involved in attenuation of PTI. Here, we describe a growth-promoting peptide hormone, phytosulfokine (PSK), as attenuating PTI signaling in Arabidopsis. This research was motivated by the observation that expression of the PSK Receptor 1 (PSKR1) gene was induced by MAMP treatment. Plants homozygous for pskr1 T-DNA insertions showed enhanced defense gene expression and seedling growth inhibition triggered by MAMPs. The pskr1 plants also showed enhanced PTI against the bacterial pathogen Pseudomonas syringae. These results indicate that the PSKR-mediated signaling attenuates immune responses. Tyrosyl protein sulfotransferase (TPST) is an enzyme required for production of the mature sulfated PSK. Like pskr1 mutants, a tpst T-DNA insertion line exhibited enhanced MAMP-triggered seedling growth inhibition, which was suppressed by exogenous application of PSK. Thus, PSK signaling mediated by PSKR1 attenuates PTI but stimulates growth.     

Kinase activity and calmodulin binding are essential for growth signaling by the phytosulfokine receptor PSKR1

The cell growth‐promoting peptide phytosulfokine (PSK) is perceived by leucine‐rich repeat (LRR) receptor kinases. To elucidate PSK receptor function we analyzed PSKR1 kinase activity and binding to Ca²⁺ sensors and evaluated the contribution of these activities to growth control in planta. Ectopically expressed PSKR1 was capable of auto‐ and transphosphorylation. Replacement of a conserved lysine within the ATP‐binding region by a glutamate resulted in the inhibition of auto‐ and transphosphorylation kinase activities. Expression of the kinase‐inactive PSKR1(K762E) receptor in the pskr null background did not restore root or shoot growth. Instead, the mutant phenotype was enhanced suggesting that the inactive receptor protein exerts growth‐inhibitory activity. Bioinformatic analysis predicted a putative calmodulin (CaM)‐binding site within PSKR1 kinase subdomain VIa. Bimolecular fluorescence complementation analysis demonstrated that PSKR1 binds to all isoforms of CaM, more weakly to the CaM‐like protein CML8 but apparently not to CML9. Mutation of a conserved tryptophan (W831S) within the predicted CaM‐binding site strongly reduced CaM binding. Expression of PSKR1(W831S) in the pskr null background resulted in growth inhibition that was similar to that of the kinase‐inactive receptor. We conclude that PSK signaling requires Ca²⁺/CaM binding and kinase activity of PSKR1 in planta. We further propose that the inactivated kinase interferes with other growth‐promoting signaling pathway(s).     

Reactive oxygen species generated in chloroplasts contribute to tobacco leaf infection by the necrotrophic fungus Botrytis cinerea

Reactive oxygen species (ROS) play fundamental roles in plant responses to pathogen infection, including modulation of cell death processes and defense‐related gene expression. Cell death triggered as part of the hypersensitive response enhances resistance to biotrophic pathogens, but favors the virulence of necrotrophs. Even though the involvement of ROS in the orchestration of defense responses is well established, the relative contribution of specific subcellular ROS sources to plant resistance against microorganisms with different pathogenesis strategies is not completely known. The aim of this work was to investigate the role of chloroplastic ROS in plant defense against a typical necrotrophic fungus, Botrytis cinerea. For this purpose, we used transgenic Nicotiana tabacum (tobacco) lines expressing a plastid‐targeted cyanobacterial flavodoxin (pfld lines), which accumulate lower chloroplastic ROS in response to different stresses. Tissue damage and fungal growth were significantly reduced in infected leaves of pfld plants, as compared with infected wild‐type (WT) counterparts. ROS build‐up triggered by Botrytis infection and associated with chloroplasts was significantly decreased (70–80%) in pfld leaves relative to the wild type. Phytoalexin accumulation and expression of pathogenesis‐related genes were induced to a lower degree in pfld plants than in WT siblings. The impact of fungal infection on photosynthetic activity was also lower in pfld leaves. The results indicate that chloroplast‐generated ROS play a major role in lesion development during Botrytis infection. This work demonstrates that the modulation of chloroplastic ROS levels by the expression of a heterologous antioxidant protein can provide a significant degree of protection against a canonical necrotrophic fungus.     

Danger peptide receptor signaling in plants ensures basal immunity upon pathogen‐induced depletion of BAK1

Pathogens infect a host by suppressing defense responses induced upon recognition of microbe‐associated molecular patterns (MAMPs). Despite this suppression, MAMP receptors mediate basal resistance to limit host susceptibility, via a process that is poorly understood. The Arabidopsis leucine‐rich repeat (LRR) receptor kinase BAK1 associates and functions with different cell surface LRR receptors for a wide range of ligands, including MAMPs. We report that BAK1 depletion is linked to defense activation through the endogenous PROPEP peptides (Pep epitopes) and their LRR receptor kinases PEPR1/PEPR2, despite critical defects in MAMP signaling. In bak1‐knockout plants, PEPR elicitation results in extensive cell death and the prioritization of salicylate‐based defenses over jasmonate‐based defenses, in addition to elevated proligand and receptor accumulation. BAK1 disruption stimulates the release of PROPEP3, produced in response to Pep application and during pathogen challenge, and renders PEPRs necessary for basal resistance. These findings are biologically relevant, since specific BAK1 depletion coincides with PEPR‐dependent resistance to the fungal pathogen Colletotrichum higginsianum. Thus, the PEPR pathway ensures basal resistance when MAMP‐triggered defenses are compromised by BAK1 depletion.     

Tryptophan‐derived secondary metabolites in Arabidopsis thaliana confer non‐host resistance to necrotrophic Plectosphaerella cucumerina fungi

A defence pathway contributing to non‐host resistance to biotrophic fungi in Arabidopsis involves the synthesis and targeted delivery of the tryptophan (trp)‐derived metabolites indol glucosinolates (IGs) and camalexin at pathogen contact sites. We have examined whether these metabolites are also rate‐limiting for colonization by necrotrophic fungi. Inoculation of Arabidopsis with adapted or non‐adapted isolates of the ascomycete Plectosphaerella cucumerina triggers the accumulation of trp‐derived metabolites. We found that their depletion in cyp79B2 cyp79B3 mutants renders Arabidopsis fully susceptible to each of three tested non‐adapted P. cucumerina isolates, and super‐susceptible to an adapted P. cucumerina isolate. This assigns a key role to trp‐derived secondary metabolites in limiting the growth of both non‐adapted and adapted necrotrophic fungi. However, 4‐methoxy‐indol‐3‐ylmethylglucosinolate, which is generated by the P450 monooxygenase CYP81F2, and hydrolyzed by PEN2 myrosinase, together with the antimicrobial camalexin play a minor role in restricting the growth of the non‐adapted necrotrophs. This contrasts with a major role of these two trp‐derived phytochemicals in limiting invasive growth of non‐adapted biotrophic powdery mildew fungi, thereby implying the existence of other unknown trp‐derived metabolites in resistance responses to non‐adapted necrotrophic P. cucumerina. Impaired defence to non‐adapted P. cucumerina, but not to the non‐adapted biotrophic fungus Erysiphe pisi, on cyp79B2 cyp79B3 plants is largely restored in the irx1 background, which shows a constitutive accumulation of antimicrobial peptides. Our findings imply differential contributions of antimicrobials in non‐host resistance to necrotrophic and biotrophic pathogens.     

The Arabidopsis LecRK‐VI.2 associates with the pattern‐recognition receptor FLS2 and primes Nicotiana benthamiana pattern‐triggered immunity

Pattern‐triggered immunity (PTI) is broad spectrum and manipulation of PTI is believed to represent an attractive way to engineer plants with broad‐spectrum disease resistance. PTI is activated upon perception of microbe‐associated molecular patterns (MAMPs) by pattern‐recognition receptors (PRRs). We have recently demonstrated that the L‐type lectin receptor kinase‐VI.2 (LecRK‐VI.2) positively regulates Arabidopsis thaliana PTI. Here we show through in vitro pull‐down, bimolecular fluorescence complementation and co‐immunoprecipitation analyses that LecRK‐VI.2 associates with the PRR FLS2. We also demonstrated that LecRK‐VI.2 from the cruciferous plant Arabidopsis remains functional after interfamily transfer to the Solanaceous plant Nicotiana benthamiana. Wild tobacco plants ectopically expressing LecRK‐VI.2 were indeed more resistant to virulent hemi‐biotrophic and necrotrophic bacteria, but not to the fungal pathogen Botrytis cinerea suggesting that, as with Arabidopsis, the LecRK‐VI.2 protective effect in N. benthamiana is bacteria specific. Ectopic expression of LecRK‐VI.2 in N. benthamiana primed PTI‐mediated reactive oxygen species production, mitogen‐activated protein kinase (MAPK) activity, callose deposition and gene expression upon treatment with the MAMP flagellin. Our findings identified LecRK‐VI.2 as a member of the FLS2 receptor complex and suggest that heterologous expression of components of PRR complexes can be used as tools to engineer plant disease resistance to bacteria.     

Insights into the role of jasmonic acid-mediated defenses against necrotrophic and biotrophic fungal pathogens

Jasmonic acid (JA) is a natural hormone regulator involved in development,responses against wounding and pathogen attack.Upon perception of pathogens,JA is synthesized and mediates a signaling cascade ...     

Silencing of OPR3 in tomato reveals the role of OPDA in callose deposition during the activation of defense responses against Botrytis cinerea

Cis‐(+)‐12‐oxo‐phytodienoic acid (OPDA) is likely to play signaling roles in plant defense that do not depend on its further conversion to the phytohormone jasmonic acid. To elucidate the role of OPDA in Solanum lycopersicum (tomato) plant defense, we have silenced the 12‐oxophytodienoate reductase 3 (OPR3) gene. Two independent transgenic tomato lines (SiOPR3‐1 and SiOPR3‐2) showed significantly reduced OPR3 expression upon infection with the necrotrophic pathogen Botrytis cinerea. Moreover, SiOPR3 plants are more susceptible to this pathogen, and this susceptibility is accompanied by a significant decrease in OPDA levels and by the production of JA‐Ile being almost abolished. OPR3 silencing also leads to a major reduction in the expression of other genes of the jasmonic acid (JA) synthesis and signaling pathways after infection. These results confirm that in tomato plants, as in Arabidopsis, OPR3 determines OPDA availability for JA biosynthesis. In addition, we show that an intact JA biosynthetic pathway is required for proper callose deposition, as its pathogen‐induced accumulation is reduced in SiOPR3 plants. Interestingly, OPDA, but not JA, treatment restored basal resistance to B. cinerea and induced callose deposition in SiOPR3‐1 and SiOPR3‐2 transgenic plants. These results provide clear evidence that OPDA by itself plays a major role in the basal defense of tomato plants against this necrotrophic pathogen.     

A constitutive PR-1::luciferase expression screen identifies Arabidopsis mutants with differential disease resistance to both biotrophic and necrotrophic pathogens

A complex signal transduction network involving salicylic acid, jasmonic acid and ethylene underlies disease resistance in Arabidopsis. To understand this defence signalling network further, we identified mutants that expressed the marker gene PR-1::luciferase in the absence of pathogen infection. These cir mutants all display constitutive expression of a suite of defence-related genes but exhibit different disease resistance profiles to two biotrophic pathogens, Pseudomonas syringae pv. tomato and Peronospora parasitica NOCO2, and the necrotrophic pathogen Botrytis cinerea. We further characterized cir3, which displays enhanced resistance only to the necrotrophic pathogen. Cir3-mediated resistance to B. cinerea is dependent on accumulated salicylic acid and a functional EIN2 protein.     

MAPK phosphatase MKP2 mediates disease responses in Arabidopsis and functionally interacts with MPK3 and MPK6

Mitogen‐activated protein kinase (MAPK) cascades have important functions in plant stress responses and development and are key players in reactive oxygen species (ROS) signalling and in innate immunity. In Arabidopsis, the transmission of ROS and pathogen signalling by MAPKs involves the coordinated activation of MPK6 and MPK3; however, the specificity of their negative regulation by phosphatases is not fully known. Here, we present genetic analyses showing that MAPK phosphatase 2 (MKP2) regulates oxidative stress and pathogen defence responses and functionally interacts with MPK3 and MPK6. We show that plants lacking a functional MKP2 gene exhibit delayed wilting symptoms in response to Ralstonia solanacearum and, by contrast, acceleration of disease progression during Botrytis cinerea infection, suggesting that this phosphatase plays differential functions in biotrophic versus necrotrophic pathogen‐induced responses. MKP2 function appears to be linked to MPK3 and MPK6 regulation, as indicated by BiFC experiments showing that MKP2 associates with MPK3 and MPK6 in vivo and that in response to fungal elicitors MKP2 exerts differential affinity versus both kinases. We also found that MKP2 interacts with MPK6 in HR‐like responses triggered by fungal elicitors, suggesting that MPK3 and MPK6 are subject to differential regulation by MKP2 in this process. We propose that MKP2 is a key regulator of MPK3 and MPK6 networks controlling both abiotic and specific pathogen responses in plants.     

The BRI1-associated kinase 1, BAK1, has a brassinolide-independent role in plant cell-death control

Programmed cell death (PCD) is a common host response to microbial infection [1-3]. In plants, PCD is associated with immunity to biotrophic pathogens, but it can also promote disease upon infection by necrotrophic pathogens [4]. Therefore, plant cell-suicide programs must be strictly controlled. Here we demonstrate that the Arabidopsis thaliana Brassinosteroid Insensitive 1 (BRI1)-associated receptor Kinase 1 (BAK1), which operates as a coreceptor of BRI1 in brassinolide (BL)-dependent plant development, also regulates the containment of microbial infection-induced cell death. BAK1-deficient plants develop spreading necrosis upon infection. This is accompanied by production of reactive oxygen intermediates and results in enhanced susceptibility to necrotrophic fungal pathogens. The exogenous application of BL rescues growth defects of bak1 mutants but fails to restore immunity to fungal infection. Moreover, BL-insensitive and -deficient mutants do not exhibit spreading necrosis or enhanced susceptibility to fungal infections. Together, these findings suggest that plant steroid-hormone signaling is dispensable for the containment of infection-induced PCD. We propose a novel, BL-independent function of BAK1 in plant cell-death control that is distinct from its BL-dependent role in plant development.