Protein Polyubiquitination Plays a Role in Basal Host Resistance of Barley

Abstract

To study protein ubiquitination pathways in the interaction of barley (Hordeum vulgare) with the powdery mildew fungus (Blumeria graminis), we measured protein turnover and performed transient-induced gene silencing (TIGS) of ubiquitin and 26S proteasome subunit encoding genes in epidermal cells. Attack by B. graminis hyperdestabilized a novel unstable green fluorescent protein fusion that contains a destabilization domain of a putative barley 1-aminocyclopropane-1-carboxylate synthase, suggesting enhanced protein turnover. Partial depletion of cellular ubiquitin levels by TIGS induced extreme susceptibility of transformed cells toward the appropriate host pathogen B. graminis f. sp hordei, whereas papilla-based resistance to the nonhost pathogen B. graminis f. sp tritici and host resistance mediated by the mlo gene (for mildew resistance locus O) remained unaffected. Cells were rescued from TIGS-induced ubiquitin depletion by synthetic genes encoding wildtype or mutant barley monoubiquitin proteins. The strongest rescue was from a gene encoding a K63R mutant form of ubiquitin blocked in several ubiquitination pathways while still allowing Lys-48-dependent polyubiquitination required for proteasomal protein degradation. Systematic RNA interference of 40 genes encoding all 17 subunits of the proteasome 19S regulatory particle failed to induce hypersusceptibility against B. graminis f. sp hordei. This suggests a role for Lys-48-linked protein polyubiquitination, which is independent from the proteasome pathway, in basal host defense of barley.

Host-Induced Gene Silencing in Barley Powdery Mildew Reveals a Class of Ribonuclease-Like Effectors

“…Nevertheless, we controlled for this possibility by assessing the ability of alternate BEC1054 and BEC1011 coding constructs with no possible targeting by the RNAi constructs (Dong et al 2006;Nowara et al 2010) to complement the phenotypes that resulted from the HIGS constructs. Co-bombardment of these wobble constructs with their respective HIGS constructs did not affect the HI, indicating that the effect of the HIGS constructs alone on HI is, indeed, dependent on the direct silencing of the respective BEC.…”

Ubiquitination during Plant Immune Signaling

“…Partial depletion of ubiquitin levels by transient-induced silencing of the ubiquitin-encoding gene in barley (Hordeum vulgare) epidermal cells resulted in enhanced susceptibility to the powdery mildew fungus Blumeria graminis f. sp. hordei (Dong et al, 2006). Moreover, complementation studies suggested a role for Lys-48-linked polyubiquitination in defense signaling (Dong et al, 2006).…”

The Multivesicular Body-Localized GTPase ARFA1b/1c Is Important for Callose Deposition and ROR2 Syntaxin-Dependent Preinvasive Basal Defense in Barley

“…Importantly, transformation of ror2 with the ARFA1b-T31N construct had no additional effect on the frequency of cells with haustoria ( Figure 3), implying that ROR2 and ARFA1b/1c act on the same pathway. To rule out the possibility that a penetration maximum already is reached in ror2 and that this is the reason why no further increase in cells with haustoria was observed after transformation with ARFA1b-T31N, we silenced polyubiquitin, previously found to dramatically affect penetration resistance in barley (Dong et al, 2006). The polyubiquitin RNAi construct increased the frequency of cells with haustoria in ror2 by 2.7-fold, showing that a penetration maximum had not been reached (Figure 3).…”

“…The Ubq RNAi construct was generated from a polyubiquitin EST sequence in the pDNR-LIB vector (BD Biosciences). The EST was derived from the same gene as HO13K06 used by Dong et al (2006). A PCR fragment of this polyubiquitin sequence, amplified using a gene-specific (Hv Ubq) and a vector-specific primer (Universal forward) (see Supplemental Table 2 online), was inserted into the entry vector pIPKTA38 according to Douchkov et al (2005).…”