Involvement of Arabidopsis thaliana endoplasmic reticulum KDEL-tailed cysteine endopeptidase 1 (AtCEP1) in powdery mildew-induced and AtCPR5-controlled cell death

Programmed cell death (PCD) is a prerequisite for successful development and it limits the spread of biotrophic pathogens in a rapid hypersensitive response at the site of infection. KDEL-tailed cysteine endopeptidases (KDEL CysEP) are a subgroup of papain-type cysteine endopeptidases expressed in tissues undergoing PCD. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2, and AtCEP3) are expressed. We have previously shown that AtCEP1 is a factor of basal resistance to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum, and is expressed in spatiotemporal association with the late fungal development on Arabidopsis leaves. The endoplasmic reticulum-localized proenzyme of AtCEP1 was further visualized at the haustorial complex encased with callose. The AtCPR5 gene (CONSTITUTIVE EXPRESSION OF PR GENES 5) is a regulator of expression of pathogenesis related genes. Loss of AtCPR5 leads to spontaneous expression of chlorotic lesions which was associated with enhanced expression of AtCEP1. We used the atcpr5-2 mutant plants and the atcep1 atcpr5-2 double mutants harboring a non-functional reporter (PCEP1:: pre-pro-3xHA-EGFP-KDEL) for visualization of AtCEP1 promoter activity. We found the specific up-regulation of AtCEP1 in direct neighborhood of spreading leaf lesions thus likely representing cells undergoing PCD. Furthermore, we found a strong resistance of atcpr5 mutant plants against infection with E. cruciferarum. Loss of AtCEP1 had no obvious influence on the strong resistance of atcpr5-2 mutant plants against infection with E. cruciferarum. However, the area of necrotic leaf lesions associated with E. cruciferarum colonies was significantly larger in atcpr5-2 as compared to atcep1 atcpr5-2 double mutant plants. The presence of AtCEP1 thus contributes to AtCPR5-controlled PCD at the sites of powdery mildew infection

Ex vivo processing for maturation of Arabidopsis KDEL-tailed cysteine endopeptidase 2 (AtCEP2) pro-enzyme and its storage in endoplasmic reticulum derived organelles

Ricinosomes are specialized ER-derived organelles that store the inactive pro-forms of KDEL-tailed cysteine endopeptidases (KDEL-CysEP) associated with programmed cell death (PCD). The Arabidopsis genome encodes three KDEL-CysEP (AtCEP1, AtCEP2, and AtCEP3) that are differentially expressed in vegetative and generative tissues undergoing PCD. These Arabidopsis proteases have not been characterized at a biochemical level, nor have they been localized intracellularly. In this study, we characterized AtCEP2. A 3xHA-mCherry-AtCEP2 gene fusion including pro-peptide and KDEL targeting sequences expressed under control of the endogenous promoter enabled us to isolate AtCEP2 "ex vivo". The purified protein was shown to be activated in a pH-dependent manner. After activation, however, protease activity was pH-independent. Analysis of substrate specificity showed that AtCEP2 accepts proline near the cleavage site, which is a rare feature specific for KDEL-CysEPs. mCherry-AtCEP2 was detected in the epidermal layers of leaves, hypocotyls and roots; in the root, it was predominantly found in the elongation zone and root cap. Co-localization with an ER membrane marker showed that mCherry-AtCEP2 was stored in two different types of ER-derived organelles: 10 μm long spindle shaped organelles as well as round vesicles with a diameter of approximately 1 μm. The long organelles appear to be ER bodies, which are found specifically in Brassicacae. The round vesicles strongly resemble the ricinosomes first described in castor bean. This study provides a first evidence for the existence of ricinosomes in Arabidopsis, and may open up new avenues of research in the field of PCD and developmental tissue remodeling.publishe

Expression analysis of KDEL-CysEPs programmed cell death markers during reproduction in Arabidopsis

Programmed cell death (PCD) is essential for proper plant growth and development. Plant-specific papain-type KDEL-tailed cysteine endopeptidases (KDEL-CysEPs or CEPs) have been shown to be involved in PCD during vegetative development as executors for the last step in the process. The Arabidopsis genome encodes three KDEL-CysEPs: AtCEP1, AtCEP2 and AtCEP3. With the help of fluorescent fusion reporter lines, we report here a detailed expression analysis of KDEL-CysEP (pro)proteins during reproductive processes, including flower organ and germline development, fertilization and seed development. AtCEP1 is highly expressed in different reproductive tissues including nucellus cells of mature ovule and the connecting edge of anther and filament. After fertilization, AtCEP1 marks integument cell layers of the seeds coat as well as suspensor and columella cells of the developing embryo. Promoter activity of AtCEP2 is detected in the style of immature and mature pistils, in other floral organs including anther, sepal and petal. AtCEP2 mainly localizes to parenchyma cells next to xylem vessels. Although there is no experimental evidence to demonstrate that KDEL-CysEPs are involved in PCD during fertilization, the expression pattern of AtCEPs, which were previously shown to represent cell death markers during vegetative development, opens up new avenues to investigate PCD in plant reproduction

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

Calmodulin-like protein AtCML3 mediates dimerization of peroxisomal processing protease AtDEG15 and contributes to normal peroxisome metabolism

Matrix enzymes are imported into peroxisomes and glyoxysomes, a subclass of peroxisomes involved in lipid mobilization. Two peroxisomal targeting signals (PTS), the C-terminal PTS1 and the N-terminal PTS2, mediate the translocation of proteins into the organelle. PTS2 processing upon import is conserved in higher eukaryotes, and in watermelon the glyoxysomal processing protease (GPP) was shown to catalyse PTS2 processing. GPP and its ortholog, the peroxisomal DEG protease from Arabidopsis thaliana (AtDEG15), belong to the Deg/HtrA family of ATP-independent serine proteases with Escherichia coli DegP as their prototype. GPP existes in monomeric and dimeric forms. Their equilibrium is shifted towards the monomer upon Ca(2+)-removal and towards the dimer upon Ca(2+)-addition, which is accompanied by a change in substrate specificity from a general protease (monomer) to the specific cleavage of the PTS2 (dimer). We describe the Ca(2+)/calmodulin (CaM) mediated dimerization of AtDEG15. Dimerization is mediated by the CaM-like protein AtCML3 as shown by yeast two and three hybrid analyses. The binding of AtCML3 occurs within the first 25 N-terminal amino acids of AtDEG15, a domain containing a predicted CaM-binding motif. Biochemical analysis of AtDEG15 deletion constructs in planta support the requirement of the CaM-binding domain for PTS2 processing. Phylogenetic analyses indicate that the CaM-binding site is conserved in peroxisomal processing proteases of higher plants (dicots, monocots) but not present in orthologs of animals or cellular slime molds. Despite normal PTS2 processing activity, an atcml3 mutant exhibited reduced 2,4-DB sensitivity, a phenotype previously reported for the atdeg15 mutant, indicating similarly impaired peroxisome metabolism.publishe

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

<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

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

The role of KDEL-tailed cysteine endopeptidases of Arabidopsis (AtCEP2 and AtCEP1) in root development.

Plants encode a unique group of papain-type cysteine endopeptidases (CysEP) characterized by a C-terminal KDEL endoplasmic reticulum retention signal (KDEL-CysEP) and an unusually broad substrate specificity. The three Arabidopsis KDEL-CysEPs (AtCEP1, AtCEP2, and AtCEP3) are differentially expressed in vegetative and generative tissues undergoing programmed cell death (PCD). While KDEL-CysEPs have been shown to be implicated in the collapse of tissues during PCD, roles of these peptidases in processes other than PCD are unknown. Using mCherry-AtCEP2 and EGFP-AtCEP1 reporter proteins in wild type versus atcep2 or atcep1 mutant plants, we explored the participation of AtCEP in young root development. Loss of AtCEP2, but not AtCEP1 resulted in shorter primary roots due to a decrease in cell length in the lateral root (LR) cap, and impairs extension of primary root epidermis cells such as trichoblasts in the elongation zone. AtCEP2 was localized to root cap corpses adherent to epidermal cells in the rapid elongation zone. AtCEP1 and AtCEP2 are expressed in root epidermis cells that are separated for LR emergence. Loss of AtCEP1 or AtCEP2 caused delayed emergence of LR primordia. KDEL-CysEPs might be involved in developmental tissue remodeling by supporting cell wall elongation and cell separation