The recognition of ubiquitylated substrates is an essential element of ubiquitin ⁄ 26S proteasome-mediated proteolysis (UPP), which is mediated directly by the proteasome subunit RPN10 and ⁄ or RPN13, or indirectly by ubiquitin receptors containing ubiquitin-like and ubiquitin-associated domains. By pull-down and mutagenesis assays, we detected cross-species divergence of the major recognition pathways. RPN10 plays a major role in direct recognition in Arabidopsis and yeast based on the strong affinity for the long and K48-linked ubiquitin chains. In contrast, both the RPN10 and RPN13 homologs play major roles in humans. For indirect recognition, the RAD23 and DSK2 homologs (except for the human DSK2 homolog) are major receptors. The human RAD23 homolog is targeted to the 26S proteasome by the RPN10 and RPN13 homologs. In comparison, Arabidopsis uses UIM1 and UIM3 of RPN10 to bind DSK2 and RAD23, respectively. Yeast uses UIM in RPN10 and LRR in RPN1. Overall, multiple proteasome subunits are responsible for the direct and ⁄ or indirect recognition of ubiquitylated substrates in yeast and humans. In contrast, a single proteasome subunit, RPN10, is critical for both the direct and indirect recognition pathways in Arabidopsis. In agreement with these results, the accumulation of ubiquitylated substrates and severe pleiotropic phenotypes of vegetative and reproductive growth are associated with the loss of RPN10 function in an Arabidopsis T-DNA insertion mutant. This implies that the targeting and proteolysis of the critical regulators involved are affected. These results support a cross-species mechanistic and functional divergence of the major recognition pathways for ubiquitylated substrates of UPP. Structured digital abstractl A list of the large number of protein-protein interactions described in this article is available via the MINT article ID MINT-7307429Abbreviations GST, glutathione S-transferase; LRR, leucine-rich repeat; PRU, Pleckstrin-like receptor of ubiquitin; RP, regulatory particle; UBA, ubiquitinassociated domain; UBL, ubiquitin-like domain; UIM, ubiquitin-interacting motif; UPP, ubiquitin ⁄ 26S proteasome-mediated proteolysis; Y2H, yeast two-hybrid analysis.

Ubiquitylated substrate recognition during ubiquitin/proteasome-mediated proteolysis (UPP) is mediated directly by the proteasome subunits RPN10 and RPN13 and indirectly by ubiquitin-like (UBL) and ubiquitin-associated (UBA) domain-containing factors. To dissect the complexity and functional roles of UPP substrate recognition in Arabidopsis thaliana, potential UPP substrate receptors were characterized. RPN10 and members of the UBL-UBA-containing RAD23 and DSK2 families displayed strong affinities for Lys-48-linked ubiquitin chains (the major UPP signals), indicating that they are involved in ubiquitylated substrate recognition. Additionally, RPN10 uses distinct interfaces as primary proteasomal docking sites for RAD23s and DSK2s. Analyses of T-DNA insertion knockout or RNA interference knockdown mutants of potential UPP ubiquitin receptors, including RPN10, RPN13, RAD23a-d, DSK2a-b, DDI1, and NUB1, demonstrated that only the RPN10 mutant gave clear phenotypes. The null rpn10-2 showed decreased double-capped proteasomes, increased 20S core complexes, and pleiotropic vegetative and reproductive growth phenotypes. Surprisingly, the observed rpn10-2 phenotypes were rescued by a RPN10 variant defective in substrate recognition, indicating that the defectiveness of RPN10 in proteasome but not substrate recognition function is responsible for the null phenotypes. Our results suggest that redundant recognition pathways likely are used in Arabidopsis to target ubiquitylated substrates for proteasomal degradation and that their specific roles in vivo require further examination.

Fifty plant extracts, four oil cakes and eight antagonistic organisms were tested against Bipolaris oryzae (Cochliobolus miyabeanus), the causal agent of brown spot disease of rice. In vitro studies indicated that two leaf extracts, Nerium oleander and Pithecolobium dulce exerted the higher percent inhibition to mycelial growth (77.4, 75.1%) and spore germination (80.3, 80.0%) of B. oryzae. Among the four oil cake extracts tested in vitro against B. oryzae, neem cake extract showed the maximum inhibition percent to mycelial growth (80.18%) and spore germination (81.13%) of the pathogen followed by mahua cake extract, castor and gingelly cake extract. Trichoderma viride (Tv2) was significantly effective in inhibiting the mycelial growth (62.92%) and spore germination (77.03%) of the pathogen followed by Trichoderma harzianum (Th5) and Trichoderma reesei (Tr3). The promising leaf extracts, oil cake extracts and antagonistic microorganisms were further evaluated for their efficacies in disease management under glasshouse and field conditions. In glasshouse studies, post-infectional spraying of rice plants with neem cake extract, N. oleander leaf extract and T. viride (Tv2) was significantly effective in reducing the incidence of brown spot of rice by 66, 52 and 45 percent respectively. Two rounds of spraying of rice plants with neem cake extract, N. oleander leaf extract and T. viride (Tv2) in the field at initial appearance of disease and 15 days later reduced the incidence of brown spot (70, 53 and 48% disease reduction respectively) and increased the yield by 23, 18 and 15 percent respectively

The reverse reaction of ubiquitylation is catalyzed by different classes of deubiquitylation enzymes (DUBs), including ovarian tumor domain (OTU)-containing DUBs; experiments using Homo sapiens proteins have demonstrated that OTU DUBs modulate various cellular processes. With the exception of OTLD1, plant OTU DUBs have not been characterized. We identified 12 Arabidopsis thaliana OTU loci and analyzed 11 of the encoded proteins in vitro to determine their preferences for the ubiquitin (UB) chains of M1, K48, and K63 linkages as well as the UB-/RUB-/SUMO-GST fusions. The A. thaliana OTU DUBs were shown to be cysteine proteases and classified into four groups with distinct linkage preferences: OTU1 (M1 = K48 > K63), OTU3/4/7/10 (K63 > K48 > M1), OTU2/9 (K48 = K63), and OTU5/11/12/OTLD1 (inactive). Five active OTU DUBs (OTU3/4/7/9/10) also cleaved RUB fusion. OTU1/3/4 cleaved M1 UB chains, suggesting a possible role for M1 chains in plant cellular signaling. The different substrate specificities of the various A. thaliana OTU DUBs indicate the involvement of distinct structural elements; for example, the OTU1 oxyanion residue D89 is essential for cleaving isopeptide bond-linked chains but dispensable for M1 chains. UB-binding activities were detected only for OTU2 and OTLD1, with distinct linkage preferences. These differences in biochemical properties support the involvement of A. thaliana OTU DUBs in different functions. Moreover, based on the established phylogenetic tree, plant- and H. sapiens-specific clades exist, which suggests that the proteins within these clades have taxa-specific functions. We also detected five OTU clades that are conserved across species, which suggests that the orthologs in different species within each clade are involved in conserved cellular processes, such as ERAD and DNA damage responses. However, different linkage preferences have been detected among potential cross-species OTU orthologs, indicating functional and mechanistic differentiation.

Phosphorus, taken up by plants as inorganic phosphate (Pi), is an essential but often growth-43 limiting mineral nutrient for plants. As part of an orchestrated response to improve its 44 acquisition, insufficient Pi supply triggers alterations in root architecture and epidermal cell 45 morphogenesis. Mutants defective in the expression of the OVARIAN TUMOR CONTAINING DEUBIQUITINATING ENZYME5 (OTU5) exhibited a constitutive Pi deficiency 47 root phenotype, comprising the formation of long and dense root hairs and attenuated primary 48 root growth. Quantitative protein profiling of otu5 and wild-type roots using the iTRAQ (isobaric 49Tag for Relative and Absolute Quantification) methodology revealed genotype-and Pi-50 dependent alterations in protein profiles. In otu5 plants, Pi starvation caused a short root hair 51 phenotype and decreased abundance of a suite of Pi-responsive root hair-related proteins. Mutant 52 plants also showed accumulation of proteins involved in chromatin remodeling and altered 53 distribution of reactive oxygen species along the root, which may be causative for the alterations 54 in root hair morphogenesis. The root hair phenotype of otu5 was synergistic to that of arp6, 55 harboring a mutation in the SWR1 chromatin remodeling complex. Genetic analysis of otu5 arp6 56 double mutants suggests independent but functionally related roles of the two proteins in 57 chromatin organization. The root hair phenotype of otu5 is not caused by a general up-regulation 58 of the Pi starvation response, indicating that OTU5 acts downstream of or is interacting with Pi 59 signaling. It is concluded that OTU5 is involved in the interpretation of environmental 60 information, probably by altering chromatin organization and maintaining redox homeostasis. 4 INTRODUCTION 62Phosphorus is an essential structural component of nucleic acids, ATP and cell membranes 63 and plays pivotal roles in signaling and catalytic reactions in metabolism. In plants, phosphorus 64 is taken up as phosphate (Pi), which has limited phyto-availability in most ecosystems and 65 agricultural settings due to complex interactions with soil constituents. Plants, in particular 66 genera that do not engage in mycorrhizal symbiosis, have evolved sophisticated strategies to 67 cope with low Pi availability involving metabolic, physiologic and developmental responses, 68 which aid in improving the acquisition, uptake, and distribution of Pi . 69Due to limited diffusion and mass flow of Pi in the soil solution, increasing the absorptive 70 surface of the root is an efficient strategy to improve Pi acquisition and uptake. In Arabidopsis, 71Pi starvation induces coordinated alterations in root architecture that comprises attenuation of 72 primary root growth and an increase in lateral root formation, a response that has been referred to 73 as topsoil foraging. The restricted longitudinal elongation of epidermal cells increases the 74 number of root hairs per unit root length in permissive positions (over anticlinal walls of cortical 75 cells;...