Endoplasmic reticulum KDEL-tailed cysteine endopeptidase 1 of Arabidopsis (AtCEP1) is involved in pathogen defense

Programmed cell death (PCD) is a genetically determined process in all multicellular organisms. Plant PCD is effected by a unique group of papain-type cysteine endopeptidases (CysEP) with a C-terminal KDEL endoplasmic reticulum (ER) retention signal (KDEL CysEP). KDEL CysEPs can be stored as pro-enzymes in ER-derived endomembrane compartments and are released as mature CysEPs in the final stages of organelle disintegration. KDEL CysEPs accept a wide variety of amino acids at the active site, including the glycosylated hydroxyprolines of the extensins that form the basic scaffold of the cell wall. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2, and AtCEP3) are expressed. Cell- and tissue-specific activities of these three genes suggest that KDEL CysEPs participate in the abscission of flower organs and in the collapse of tissues in the final stage of PCD as well as in developmental tissue remodeling. We observed that AtCEP1 is expressed in response to biotic stress stimuli in the leaf. atcep1 knockout mutants showed enhanced susceptibility to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum. A translational fusion protein of AtCEP1 with a three-fold hemaglutinin-tag and the green fluorescent protein under control of the endogenous AtCEP1 promoter (PCEP1::pre-pro-3xHA-EGFP-AtCEP1-KDEL) rescued the pathogenesis phenotype demonstrating the function of AtCEP1 in restriction of powdery mildew. The spatiotemporal AtCEP1-reporter expression during fungal infection together with microscopic inspection of the interaction phenotype suggested a function of AtCEP1 in controlling late stages of compatible interaction including late epidermal cell death. Additionally, expression of stress response genes appeared to be deregulated in the interaction of atcep1 mutants and E. cruciferarum. Possible functions of AtCEP1 in restricting parasitic success of the obligate biotrophic powdery mildew fungus are discussed.publishe

A Barley ROP GTPase ACTIVATING PROTEIN Associates with Microtubules and Regulates Entry of the Barley Powdery Mildew Fungus into Leaf Epidermal Cells[C][W]

Little is known about the role the cytoskeleton plays in signaling with regard to the outcome of plant–microbe interactions. This work describes a regulator of cytoskeleton organization and of disease susceptibility shuttling between microtubules and the monomeric G-protein RACB at the cell periphery

Three-dimensional reconstruction of fluorescence signals from the functional proenzyme of CEP1 (green) and callose depositions (blue) in epidermal cells of <i>Arabidopsis</i> invaded by haustoria of <i>E</i>.<i>cruciferarum</i>.

<p>(A) Overview about several interaction sites with callose-encased haustorial complexes in the epidermis of Arabidopsis leaves at 11 dpi. CEP1 proenzyme is visible in epidermal cells and in and around encased haustoria that are shown in an optical section and reconstructed in 3 dimensions for lower z-sections. (B-G) Accumulation of the CEP1 proenzyme at the side and within a haustorium that is encased with callose. The same haustorium is also highlighted with an arrow in A. (B) View from the cell surface. At this site, little CEP1-loaded ER is visible at the periclinal cell cortex (arrowheads) (C) View from the side of the haustorium. Arrowheads indicate the rim of the encasement closest to the periclinal cell cortex (D) Bottom up view. Arrowhead indicates incomplete closure of the callose encasement (E-G) Callose-encased haustorium cut at the line indicated in panel C viewed from different angels. Arrowhead indicates incomplete closure of the callose encasement. Black arrows indicate sites where CEP1 signals are visible at both the cytosolic and the haustorial side of the encasement. Original pictures were recorded by CLSM and reconstructed into a 3-demensional picture by the IMARIS software (Bitplane).</p

<i>cpr5-2</i> mutants are powdery mildew resistant.

<p>Disease symptoms of wild type, <i>cep1</i> and <i>cpr5-2</i> single mutants and <i>cep1 cpr5-2</i> double mutant plants upon leaf infection with <i>E</i>. <i>cruciferarum</i> were scored after visual inspection of the whole plant 10, 12 and 14 days post inoculation (dpi). Distribution of infected leaves in the three categories was carried out as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183870#pone.0183870.ref018" target="_blank">18</a>]. Infected leafs were distributed in the three categories <30%, 30–60%, and >60% diseased leaf area [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183870#pone.0183870.ref018" target="_blank">18</a>]. Columns of the same colour marked with different letters indicate statistically different groups (<i>p</i> < 0.05), and columns marked with identical letters indicate groups not statistically different (<i>p</i> > 0.05) according to the ANOVA- and Duncan test, and represent the frequency of plants distributed in the three categories of susceptibility. Data represent the respective means of eight experiments from independent inoculation events of the mutants with the corresponding parent background control. Each experiment comprised five plants per line.</p

Powdery mildew disease phenotypes caused by the biotrophic ascomycete <i>Erysiphe cruciferarum</i>.

<p>(A) <i>cep1</i> knockout mutants developed the usual symptoms upon infection with powdery mildew (arrows). (B and C) In contrast, the <i>cep1 cpr5-2</i> double mutant plants and the <i>cpr5</i> single mutant plants are resistant to <i>E</i>. <i>cruciferarum</i>; necrotic lesions are indicated by white arrow heads; please note the scaling bar: <i>cpr5</i> and <i>cep1 cpr5-2</i> mutants are dwarf. Plants in (A-C) are shown 11 days post low density inoculation. (D-F) Non-infected plants at the time of inoculation.</p

ROPGAPs of Arabidopsis limit susceptibility to powdery mildew

The barley ROP GTPase HvRACB is a susceptibility factor of barley to powdery mildew caused by the biotrophic fungus Blumeria graminis f.sp. hordei (Bgh). In a recent publication, we reported about a MICROTUBULE-ASSOCIATED ROP GTPASE-ACTIVATING PROTEIN 1 (HvMAGAP1) of barley. Transient-induced gene silencing or overexpression of HvMAGAP1 resulted in enhanced or reduced susceptibility to Bgh, respectively, indicating a possible HvRACB-antagonistic function of HvMAGAP1 in interaction with Bgh. HvMAGAP1 also influences the polarity of cortical microtubules in interaction with Bgh. In AtROPGAP1 and AtROPGAP4, Arabidopsis homologs of HvMAGAP1, knock-out T-DNA insertions enhanced susceptibility of Arabidopsis to the virulent powdery mildew fungus Erysiphe cruciferarum, indicating functions of ROPGAPs in pathogen interaction of monocots and dicots. Here we discuss the role of AtROPGAP1 and AtROPGAP4 in Arabidopsis pathogenesis of powdery mildew in some more detail