The SUMO Conjugation Complex Self-Assembles into Nuclear Bodies Independent of SIZ1 and COP1.

Attachment of the small ubiquitin-like modifier (SUMO) to substrate proteins modulates their turnover, activity, or interaction partners. However, how this SUMO conjugation activity concentrates the proteins involved and the substrates into uncharacterized nuclear bodies (NBs) remains poorly understood. Here, we characterized the requirements for SUMO NB formation and for their subsequent colocalization with the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a master regulator of plant growth. COP1 activity results in degradation of transcription factors, which primes the transcriptional response that underlies elongation growth induced by darkness and high ambient temperatures (skoto- and thermomorphogenesis, respectively). SUMO conjugation activity alone was sufficient to target the SUMO machinery into NBs. Colocalization of these bodies with COP1 required, in addition to SUMO conjugation activity, a SUMO acceptor site in COP1 and the SUMO E3 ligase SAP and Miz 1 (SIZ1). We found that SIZ1 docks in the substrate-binding pocket of COP1 via two valine-proline peptide motifs, which represent a known interaction motif of COP1 substrates. The data reveal that SIZ1 physically connects COP1 and SUMO conjugation activity in the same NBs that can also contain the blue-light receptors CRYPTOCHROME 1 and CRYPTOCHROME 2. Our findings thus suggest that sumoylation stimulates COP1 activity within NBs. Moreover, the presence of SIZ1 and SUMO in these NBs explains how both the timing and amplitude of the high-temperature growth response is controlled. The strong colocalization of COP1 and SUMO in these NBs might also explain why many COP1 substrates are sumoylated.

Experimental and bioinformatic characterization of a recombinant polygalacturonase-inhibitor protein from pearl millet and its interaction with fungal polygalacturonases.

Polygalacturonases (PGs) are hydrolytic enzymes employed by several phytopathogens to weaken the plant cell wall by degrading homopolygalacturonan, a major constituent of pectin. Plants fight back by employing polygalacturonase-inhibitor proteins (PGIPs). The present study compared the inhibition potential of pearl millet PGIP (Pennisetum glaucum; PglPGIP1) with the known inhibition of Phaseolus vulgaris PGIP (PvPGIP2) against two PGs, the PG-II isoform from Aspergillus niger (AnPGII) and the PG-III isoform from Fusarium moniliforme (FmPGIII). The key rationale was to elucidate the relationship between the extent of sequence similarity of the PGIPs and the corresponding PG inhibition potential. First, a pearl millet pgip gene (Pglpgip1) was isolated and phylogenetically placed among monocot PGIPs alongside foxtail millet (Setaria italica). Upstream sequence analysis of Pglpgip1 identified important cis-elements responsive to light, plant stress hormones, and anoxic stress. PglPGIP1, heterologously produced in Escherichia coli, partially inhibited AnPGII non-competitively with a pH optimum between 4.0 and 4.5, and showed no inhibition against FmPGIII. Docking analysis showed that the concave surface of PglPGIP1 interacted strongly with the N-terminal region of AnPGII away from the active site, whereas it weakly interacted with the C-terminus of FmPGIII. Interestingly, PglPGIP1 and PvPGIP2 employed similar motif regions with few identical amino acids for interaction with AnPGII at non-substrate-binding sites; however, they engaged different regions of AnPGII. Computational mutagenesis predicted D126 (PglPGIP1)-K39 (AnPGII) to be the most significant binding contact in the PglPGIP1-AnPGII complex. Such protein-protein interaction studies are crucial in the future generation of designer host proteins for improved resistance against ever-evolving pathogen virulence factors.

Association between accumulation of allene oxide synthase activity and development of resistance against downy mildew disease of pearl millet.

The present study was aimed at understanding the possible association of allene oxide synthase (AOS), an enzyme implicated in the octadecanoid pathway during the pearl millet-downy mildew interaction. AOS 13-HPOT (13-hydroperoxy-9,11,15-octadecatrienoic acid) metabolizing activity assays assessed in various pearl millet cultivars with differential resistances against downy mildew revealed a positive correlation between cultivar resistance levels and AOS activities. Furthermore, the involvement of AOS in response to downy mildew was demonstrated by induction of AOS activity in both susceptible and resistant pearl millet cultivars during Sclerospora graminicola infection with higher induction observed in the resistant cultivar. Consistently, western blot analysis and tissue-blot immunoassay demonstrated the remarkable increase in AOS protein accumulation in the incompatible interaction. In addition, the tissue-blot immunoassay also showed the compartmentalization of AOS in the epidermis and vascular bundles of pearl millet seedlings. Expression analysis of a putative PgAOS1 gene revealed a marked difference in accumulation of PgAOS1 transcripts between contrasting plants, with pathogen-induced higher accumulation of the transcripts observed only in the resistant cultivar; a result which is in agreement with pathogen-induced AOS level and activity, indicating that PgAOS1 plays an important role in regulation of AOS level and activity in pearl millet upon S. graminicola infection. Our findings suggest an important role for AOS in regulation of responses to downy mildew disease in pearl millet. The differential AOS activities can potentially be used for selection of new disease-resistant pearl millet varieties, and the identified AOS-encoding gene(s) as genetic resource for development of enhanced downy mildew-resistant cultivars.

Arabidopsis small ubiquitin-like modifier paralogs have distinct functions in development and defense.

Posttranslational modifications allow dynamic and reversible changes to protein function. In Arabidopsis thaliana, a small gene family encodes paralogs of the small ubiquitin-like posttranslational modifier. We studied the function of these paralogs. Single mutants of the SUM1 and SUM2 paralogs do not exhibit a clear phenotype. However, the corresponding double knockdown mutant revealed that SUM1 and SUM2 are essential for plant development, floral transition, and suppression of salicylic acid (SA)-dependent defense responses. The SUM1 and SUM2 genes are constitutively expressed, but their spatial expression patterns do not overlap. Tight transcriptional regulation of these two SUM genes appears to be important, as overexpression of either wild-type or conjugation-deficient mutants resulted in activation of SA-dependent defense responses, as did the sum1 sum2 knockdown mutant. Interestingly, expression of the paralog SUM3 is strongly and widely induced by SA and by the defense elicitor Flg22, whereas its expression is otherwise low and restricted to a few specific cell types. Loss of SUM3 does not result in an aberrant developmental phenotype except for late flowering, while SUM3 overexpression causes early flowering and activates plant defense. Apparently, SUM3 promotes plant defense downstream of SA, while SUM1 and SUM2 together prevent SA accumulation in noninfected plants.

Role of hydroxyproline-rich glycoproteins in resistance of pearl millet against downy mildew pathogen Sclerospora graminicola.

Hydroxyproline-rich glycoproteins (HRGPs) are important plant cell wall components involved in plant defense response to pathogen attack. In the present study, a resistant pearl millet (Pennisetum glaucum) cultivar, IP18292, was compared with a susceptible cultivar, 7042S, to investigate the contribution of HRGPs in the successful defense against the phytopathogenic oomycete S. graminicola. Northern hybridization using MeHRGP cDNA, a heterologous probe from cassava, indicated steady accumulation of HRGP transcripts, from 2 h.p.i. onwards with a maximum at 6 h.p.i., in the resistant cultivar. This is followed by HRGPs accumulation at about 8 h.p.i. as revealed by Western-blot analysis. Immunocytochemical localization by tissue printing and confocal immunofluorescence microscopy indicated cell walls of parenchymatic cells and the vascular tissue of coleoptile as sites of HRGP deposition. In vitro studies in the presence of horseradish peroxidase and H2O2 showed cross-linking of pearl millet HRGPs, which occurred parallel to isodityrosine accumulation. Inducible high isodityrosine content was also observed in vivo in the resistant cultivar. Here, H2O2 was found to accumulate as twin burst at 1 and 6 h.p.i., whereas in the susceptible cultivar only an early single peak was detectable. Moreover, the amount of hydroxyproline in HRGPs was about twice as high in the resistant as in the susceptible cultivar. These results suggest that cell wall strengthening in S. graminicola-infected resistant pearl millet is brought about by a combination of polypeptide cross-linking of isodityrosine as well as by the high content of hydroxyproline in HRGPs, and H2O2, in contrast to the susceptible plant.

Purification and characterization of proline/hydroxyproline-rich glycoprotein from pearl millet coleoptiles infected with downy mildew pathogen Sclerospora graminicola.

Hydroxyproline-rich glycoproteins (HRGPs) are important plant cell wall structural components, which are also involved in response to pathogen attack. In pearl millet, deposition and cross-linking of HRGPs in plant cell walls was shown to contribute to the formation of resistance barriers against the phytopathogenic oomycete Sclerospora graminicola. In the present study, the purification and characterization of HRGPs that accumulated in coleoptiles of pearl millet seedlings in response to S. graminicola inoculation has been carried out. Periodic acid Schiff's staining revealed that the purified protein was a glycoprotein. The protein to carbohydrate ratio was determined to be 95.5%:4.5% (w/w). Proline amounted for 20 mol% of the total amino acids as indicated by amino acid composition analysis. The isolated protein had a pI of 9.8 and was shown to be composed of subunits of 27, 17, and 14 kDa. Cross reactivity with the monoclonal antibody MAC 265 and the presence of the signature amino acid sequence, PVYK, strongly suggested to classify the purified glycoprotein as a member of the P/HRGPs class. In the presence of horseradish peroxidase and H2O2 the purified glycoprotein served as a substrate for oxidative cross-linking processes.