Proteomic Analysis of Post-pollination Senescence in Petunia Flowers

The senescence of vegetative and floral tissues can have a detrimentalimpact on the quality and subsequent value of agricultural and horticultural crops. To global analyses of protein expression during flower senescence, we are therefore using a proteomic approach to identify components of the senescence program in Petunia x hybrida cv Mitchell Diploid flowers. Total soluble proteins were extracted from petunia corollas at 24, 48, and 72 hours after flower opening (i.e. unpollinated, nonsenescing flowers) and at 24, 48, and 72 h after pollination (i.e. senescing flowers). Two- dimensional gel electrophoresis (2DE) was used to identify those proteins that were differentially expressed in nonsenescing (unpollinated) and senescing(pollinated) corollas. PDQuest image analysis (BioRad) software was used to identify those proteins up or down regulated by two fold in pollinated corollas. One hundred forty differentially expression proteins were identified. Most of these were identified by comparing 72 h unpollinated to 72 h pollinated corollas. LC-tandem mass spectrometry (LC-tandem MS) was used to determine the identity of these proteins. Searching the NCBI nonredundant protein and petunia translated EST database we have been able to assign a putative identification to greater than 90% of these proteins. Identified proteins are involved in many metabolic pathways including proteolysis; nuclei acid, cell wall and lipid catabolism; and signal transduction. To further characterize the role of these proteins in flower senescence, we will knockdown the expression of the corresponding genes using virus-induced gene silencing.OARDC Research Enhancement Competitive GrantThe Fred C. Gloeckner FoundationThe Ohio State University D.C. Kiplinger Endowmen

Proteomic analysis of pollination-induced corolla senescence in petunia

Senescence represents the last phase of petal development during which macromolecules and organelles are degraded and nutrients are recycled to developing tissues. To understand better the post-transcriptional changes regulating petal senescence, a proteomic approach was used to profile protein changes during the senescence of Petunia×hybrida ‘Mitchell Diploid’ corollas. Total soluble proteins were extracted from unpollinated petunia corollas at 0, 24, 48, and 72 h after flower opening and at 24, 48, and 72 h after pollination. Two-dimensional gel electrophoresis (2-DE) was used to identify proteins that were differentially expressed in non-senescing (unpollinated) and senescing (pollinated) corollas, and image analysis was used to determine which proteins were up- or down-regulated by the experimentally determined cut-off of 2.1-fold for P <0.05. One hundred and thirty-three differentially expressed protein spots were selected for sequencing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the identity of these proteins. Searching translated EST databases and the NCBI non-redundant protein database, it was possible to assign a putative identification to greater than 90% of these proteins. Many of the senescence up-regulated proteins were putatively involved in defence and stress responses or macromolecule catabolism. Some proteins, not previously characterized during flower senescence, were identified, including an orthologue of the tomato abscisic acid stress ripening protein 4 (ASR4). Gene expression patterns did not always correlate with protein expression, confirming that both proteomic and genomic approaches will be required to obtain a detailed understanding of the regulation of petal senescence

Building a better antibody through the Fc: advances and challenges in harnessing antibody Fc effector functions for antiviral protection

Antibodies can provide antiviral protection through neutralization and recruitment of innate effector functions through the Fc domain. While neutralization has long been appreciated for its role in antibody-mediated protection, a growing body of work indicates that the antibody Fc domain also significantly contributes to antiviral protection. Recruitment of innate immune cells such as natural killer cells, neutrophils, monocytes, macrophages, dendritic cells and the complement system by antibodies can lead to direct restriction of viral infection as well as promoting long-term antiviral immunity. Monoclonal antibody therapeutics against viruses are increasingly incorporating Fc-enhancing features to take advantage of the Fc domain, uncovering a surprising breadth of mechanisms through which antibodies can control viral infection. Here, we review the recent advances in our understanding of antibody-mediated innate immune effector functions in protection from viral infection and review the current approaches and challenges to effectively leverage innate immune cells via antibodies

The Senescence-Associated Endonuclease, PhENDO1, Is Upregulated by Ethylene and Phosphorus Deficiency in Petunia

The upregulation of endonuclease activities and subsequent decreases in the nucleic acid content of leaves and petals are characteristics of senescence that allow for nutrient remobilization from dying organs. We previously identified a 43-kDa endonuclease activity (PhNUC1) that was upregulated in Petunia × hybrida petals during senescence. PhNUC1 has optimal activity at neutral pH, is enhanced by Co2+, and degrades both DNA and RNA. The peptide sequence of a 43-kDa endonuclease identified from senescing petals by 2-dimensional gel electrophoresis was used to clone the gene (PhENDO1) encoding the senescence-associated protein. PhENDO1 expression was upregulated in petals during the senescence of unpollinated and pollinated flowers and by ethylene treatment. When petunias were grown under nutrient deficient conditions, P-starvation, and to a lesser extent N-starvation, induced expression of PhENDO1. The endogenous expression of PhENDO1 was down regulated using virus induced gene silencing (VIGS), and in-gel endonuclease assays confirmed that the activity of the 43-kDa PhNUC1 was decreased in senescing corollas from PhENDO1-silenced (pTRV2:PhCHS:PhENDO1) plants compared to controls (pTRV2:PhCHS). Down regulating PhENDO1 in petunias did not alter flower longevity. While PhENDO1 may be involved in nucleic acid catabolism during senescence, down regulating this gene using VIGS was not sufficient to delay flower senescence

Modelled co-structure of Khosta 2 RBD and human ACE2.

(A) Crystal structure of SARS-CoV RBD bound to human ACE2 (PDB ID: 2AJF) with contact points indicated in light blue. (B) Crystal structure of SARS-CoV-2 RBD bound to human ACE2 (PDB ID: 6M0J) with contact points indicated in light blue. (C) Predicted structure of Khosta 2 RBD bound to human ACE2 with contact points identical to either SARS-CoV or SARS-CoV-2 spike indicated in light green and resides that are different indicated in red. (D) ACE2-contact point comparison between SARS-CoV, SARS-CoV-2 and Khosta 2 spike. Residues that are identical between Khosta 2 and SARS-CoV or SARS-CoV-2 are shaded in light green.</p

Chimeric SARS-CoV-2-Khosta 2 spike is resistant to current vaccines.

(A) The RBD from SARS-Cov-2 spike was replaced with Khosta 2 RBD. (B) Pseudotpyes with indicated chimeric spikes were used to infect 293T cells stably expressing human ACE2. Pseudotypes were combined with (C) bamlanivimab or (D) vaccinated patient serum at various concentrations and used to infect 293T-hACE2 cells.(E) Area under the curve analysis for data in panel D. p-value 0.0021 (**), 0.0002 (***), F) Pseudotypes with Khosta 2 or Omicron variant RBD were combined with serum from vaccinated patients with breakthrough Omicron infection and used to infect 293T-hACE2 cells. (G) Area under the curve analysis for data in panel F. p-value 0.0021 (**), 0.0002 (***), H) Sequence identity matrix for RBD amino acid sequences from Khosta 2 and known SARS-CoV-2 variants of concern.</p

Khosta 2 and other RBD clade 3 sarbecoviruses use human ACE2 to infect cells.

(A) Sarbecovirus Receptor Binding Domain Cladogram based on amino acid sequences and rooted at the midpoint. Countries of origin and known host receptors are indicated to the right. Clade 1 viruses are shown in red and orange, clade 2 in grey, clade 3 in blue and clade 4 in purple. (B) Diagram of spike constructs used for this study. The SARS-CoV-1 RBD was replaced with RBDs from other sarbecoviruses. (C) Expression and incorporation of viral pseudotypes by westernblot. (D) Huh-7 cells were infected with pseudotypes in the presence of absence of trypsin. Cells were infected in triplicate. (E) BHK cells were transfected with receptors and infected in the absence (F) or presence of trypsin. (G) 293T cells stably over-expressing human ACE2 were infected with pseudotypes without trypsin. Cells were infected in triplicate or quadruplicate for experiments in D-G.</p

Excel file with all graphed data points from main text figures.

(XLSX)</p

Sarbecovirus sequences used in this study.

Sarbecovirus sequences used in this study.</p