Involvement of the glutamate receptor AtGLR3.3 in plant defense signaling and resistance to Hyaloperonospora arabidopsidis

Like their animal counterparts, plant glutamate receptor‐like (GLR) homologs are intimately associated with Ca²⁺ influx through plasma membrane and participate in various physiological processes. In pathogen‐associated molecular patterns (PAMP)‐/elicitor‐mediated resistance, Ca²⁺ fluxes are necessary for activating downstream signaling events related to plant defense. In this study, oligogalacturonides (OGs), which are endogenous elicitors derived from cell wall degradation, were used to investigate the role of Arabidopsis GLRs in defense signaling. Pharmacological investigations indicated that GLRs are partly involved in free cytosolic [Ca²⁺] ([Ca²⁺]_cyt) variations, nitric oxide (NO) production, reactive oxygen species (ROS) production and expression of defense‐related genes by OGs. In addition, wild‐type Col‐0 plants treated with the glutamate‐receptor antagonist 6,7‐dinitriquinoxaline‐2,3‐dione (DNQX) had a compromised resistance to Botrytis cinerea and Hyaloperonospora arabidopsidis. Moreover, we provide genetic evidence that AtGLR3.3 is a key component of resistance against H. arabidopsidis. In addition, some OGs‐triggered immune events such as defense gene expression, NO and ROS production are also to different extents dependent on AtGLR3.3. Taken together, these data provide evidence for the involvement of GLRs in elicitor/pathogen‐mediated plant defense signaling pathways in Arabidopsis thaliana.     

A PCR assay for the quantification of growth of the oomycete pathogen Hyaloperonospora arabidopsidis in Arabidopsis thaliana

The accurate quantification of disease severity is important for the assessment of host–pathogen interactions in laboratory or field settings. The interaction between Arabidopsis thaliana and its naturally occurring downy mildew pathogen, Hyaloperonospora arabidopsidis (Hpa), is a widely used reference pathosystem for plant–oomycete interactions. Current methods for the assessment of disease severity in the Arabidopsis–Hpa interaction rely on measurements at the terminal stage of pathogen development; namely, visual counts of spore‐producing structures or the quantification of spore production with a haemocytometer. These assays are useful, but do not offer sensitivity for the robust quantification of small changes in virulence or the accurate quantification of pathogen growth prior to the reproductive stage. Here, we describe a quantitative real‐time polymerase chain reaction (qPCR) assay for the monitoring of Hpa growth in planta. The protocol is rapid, inexpensive and can robustly distinguish small changes in virulence. We used this assay to investigate the dynamics of early Hpa mycelial growth and to demonstrate the proof of concept that this assay could be used in screens for novel oomycete growth inhibitors.     

Tête à tête inside a plant cell: establishing compatibility between plants and biotrophic fungi and oomycetes

'Compatibility' describes the complementary relationship between a plant species and an adapted pathogen species that underlies susceptibility and which ultimately results in disease. Owing to elaborate surveillance systems and defence mechanisms on the plant side and a common lack of adaptation of many microbial pathogens, resistance is the rule and compatibility the exception for most plant-microbe combinations. While there has been major scientific interest in 'resistance' in the past decade, which has revealed many of its underlying molecular components, the analysis of 'compatibility', although intimately intertwined with 'resistance', has not been pursued with a similar intensity. Various recent studies, however, provide a first glimpse of the pivotal players and potential molecular mechanisms essential for compatibility in both the plant and parasite partners. In this review we highlight these findings with a particular emphasis on obligate biotrophic and hemibiotrophic fungal and oomycete pathogens and discuss novel strategies that might help to uncover further the molecular principles underlying compatibility to these highly specialized pathogens.     

Probing formation of cargo/importin‐α transport complexes in plant cells using a pathogen effector

Importin‐αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin‐α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin‐α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin‐α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co‐opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin‐α paralogs from Arabidopsis thaliana. A crystal structure of the importin‐α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin‐αs expressed in rosette leaves have an almost identical NLS‐binding site. Comparison of the importin‐α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin‐α, sequence variation at the importin‐α NLS‐binding sites and tissue‐specific expression levels of importin‐αs determine formation of cargo/importin‐α transport complexes in plant cells.     

Role of the penetration‐resistance genes PEN1, PEN2 and PEN3 in the hypersensitive response and race‐specific resistance in Arabidopsis thaliana

Plants are highly capable of recognizing and defending themselves against invading microbes. Adapted plant pathogens secrete effector molecules to suppress the host's immune system. These molecules may be recognized by host‐encoded resistance proteins, which then trigger defense in the form of the hypersensitive response (HR) leading to programmed cell death of the host tissue at the infection site. The three proteins PEN1, PEN2 and PEN3 have been found to act as central components in cell wall‐based defense against the non‐adapted powdery mildew Blumeria graminis fsp. hordei (Bgh). We found that loss of function mutations in any of the three PEN genes cause decreased hypersensitive cell death triggered by recognition of effectors from oomycete and bacterial pathogens in Arabidopsis. There were considerable additive effects of the mutations. The HR induced by recognition of AvrRpm1 was almost completely abolished in the pen2 pen3 and pen1 pen3 double mutants and the loss of cell death could be linked to indole glucosinolate breakdown products. However, the loss of the HR in pen double mutants did not affect the plants' ability to restrict bacterial growth, whereas resistance to avirulent isolates of the oomycete Hyaloperonospora arabidopsidis was strongly compromised. In contrast, the double and triple mutants demonstrated varying degrees of run‐away cell death in response to Bgh. Taken together, our results indicate that the three genes PEN1, PEN2 and PEN3 extend in functionality beyond their previously recognized functions in cell wall‐based defense against non‐host pathogens.