Abstract
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.
Highlights
Programmed cell death (PCD) is a genetically determined, highly regulated process in all multicellular organisms and a prerequisite for successful development
We previously described that cells attacked by Erysiphe cruciferarum express the proenzyme of CEP1 from the functional reporter PCEP1::pre-pro-3xHA-EGFP-AtCEP1-KDEL in the endoplasmic reticulum (ER) and accumulate it around haustoria
Three dimensional reconstructions of fluorescence signals recorded by confocal laser scanning microscopy (CLSM) suggested that the proenzyme of CEP1 is closely associated with calloseencased haustoria and can be even found within encasements of haustorial complexes (Fig 1A)
Summary
Programmed cell death (PCD) is a genetically determined, highly regulated process in all multicellular organisms and a prerequisite for successful development. A unique group of papain-type cysteine endopeptidases (CysEPs) is specific for plant PCD and characterized by a C-terminal KDEL endoplasmic reticulum (ER) retention signal (KDEL CysEPs) with RcCysEP from castor bean (Ricinus communis) as the founding member [8,9,10]. KDEL CysEPs can be stored as enzymatically inactive pro-enzymes in ERderived compartments [10,12,13,14]; upon acidification, the KDEL CysEPs are released and the pro-sequence together with the C-terminal KDEL endoplasmtic reticulum retention signal are removed for activation of the enzyme [10,15] (as described in detail previously [13]). The broad substrate specificity is due to the active site cleft of the KDEL CysEPs that accepts a wide variety of amino acids including proline and glycosylated hydroxyproline of the hydroxyproline rich glycoproteins of the cell wall [17]. The respective amino acids, which are decisive for this generally more open appearance of the active site cleft, together with the amino acids defining the catalytic pocket are highly conserved among all known KDEL CysEPs [11]
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