Serological methods are relatively convenient and simple for the detection of pathogens for front-line workers. On-site visualization of the test results plays a pivotal role in the process. However, an efficient, universal labeling agent for antibodies is needed for the development of efficient serological detection tools. In this study, a Bamboo mosaic virus (BaMV)-based viral vector was employed to express recombinant proteins, collectively designated GfED, consisting of Staphylococcus aureus Protein A domain ED (SpaED) fused to either the N- or C-terminal of an improved green florescent protein (GFP) with or without the coat protein (CP) of BaMV, efficiently in Chenopodium quinoa. The GfED in crude leaf extracts could specifically attach to IgG molecules of rabbits and mice, effectively labeling IgG with GFP, emitting green light at 506 nm when excited at 450 nm using simple, handheld equipment. To demonstrate the applicability of GfED in serological assays, we have developed a fluorescent dot blot assay for the rapid detection of Acidovorax citrulli (Ac), a bacterial pathogen of cucurbits, and BaMV, a viral pathogen of bamboos. By using the crude extracts of inoculated C. quinoa leaves expressing GfED as an IgG-labeling agent, the pathogens were easily and quickly detected through uncomplicated operations using simple equipment, with results observable by the naked eye. Examination using fluorescent microscopy and transmission electron microscopy revealed that the GfED subunits may assemble into virus-like particles, which were further involved in the formation of aggregates of GfED-antibody-antigen complexes with the potential for fluorescence signal enhancement. The results suggested that plant-expressed GfED may serve as a promising alternative of IgG-labeling agent for current serological assays.

Plants are vulnerable to the challenges of unstable environments and pathogen infections due to their immobility. Among various stress conditions, viral infection is a major threat that causes significant crop loss. In response to viral infection, plants undergo complex molecular and physiological changes, which trigger defense and morphogenic pathways. Transcription factors (TFs), and their interactions with cofactors and cis-regulatory genomic elements, are essential for plant defense mechanisms. The transcriptional regulation by TFs is crucial in establishing plant defense and associated activities during viral infections. Therefore, identifying and characterizing the critical genes involved in the responses of plants against virus stress is essential for the development of transgenic plants that exhibit enhanced tolerance or resistance. This article reviews the current understanding of the transcriptional control of plant defenses, with a special focus on NAC, MYB, WRKY, bZIP, and AP2/ERF TFs. The review provides an update on the latest advances in understanding how plant TFs regulate defense genes expression during viral infection.

Plant ARGONAUTES (AGOs) play a significant role in the defense against viral infection. Previously, we have demonstrated that AGO5s encoded in Phalaenopsis aphrodite subsp. formosana (PaAGO5s) took an indispensable part in defense against major viruses. To understand the underlying defense mechanism, we cloned PaAGO5s promoters (pPaAGO5s) and analyzed their activity in transgenic Nicotiana benthamiana using β-glucuronidase (GUS) as a reporter gene. GUS activity analyses revealed that during Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) infections, pPaAGO5b activity was significantly increased compared to pPaAGO5a and pPaAGO5c. Analysis of pPaAGO5b 5′-deletion revealed that pPaAGO5b_941 has higher activity during virus infection. Further, yeast one-hybrid analysis showed that the transcription factor NbMYB30 physically interacted with pPaAGO5b_941 to enhance its activity. Overexpression and silencing of NbMYB30 resulted in up- and downregulation of GUS expression, respectively. Exogenous application and endogenous measurement of phytohormones have shown that methyl jasmonate and salicylic acid respond to viral infections. NbMYB30 overexpression and its closest related protein, PaMYB30, in P. aphrodite subsp. formosana reduced CymMV accumulation in P. aphrodite subsp. formosana. Based on these discoveries, this study uncovers the interaction between virus-responsive promoter and the corresponding transcription factor in plants.

Orchidaceae is considered the largest vascular plant family, representing more than 35,000 species in about 880 genera (Wang et al., 2016). Orchids are among the most valuable ornamental crops in horticulture and the floral industry. However, cultivation and marketability of orchids have been greatly hampered by various pathogens, especially viruses that are not effectively controlled by pesticide applications. Orchids have been reported to be infected by more than 50 species of viruses, among which Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are two of the most prevalent viral pathogens that have posed serious threats to the orchid industry.

Synergistic interactions among viruses, hosts and/or transmission vectors during mixed infection can alter viral titers, symptom severity or host range. Viral suppressors of RNA silencing (VSRs) are considered one of such factors contributing to synergistic responses. Odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV), which are two of the most significant orchid viruses, exhibit synergistic symptom intensification in Phalaenopsis orchids with unilaterally enhanced CymMV movement by ORSV. In order to reveal the underlying mechanisms, we generated infectious cDNA clones of ORSV and CymMV isolated from Phalaenopsis that exerted similar unilateral synergism in both Phalaenopsis orchid and Nicotiana benthamiana. Moreover, we show that the ORSV replicase P126 is a VSR. Mutagenesis analysis revealed that mutation of the methionine in the carboxyl terminus of ORSV P126 abolished ORSV replication even though some P126 mutants preserved VSR activity, indicating that the VSR function of P126 alone is not sufficient for viral replication. Thus, P126 functions in both ORSV replication and as a VSR. Furthermore, P126 expression enhanced cell-to-cell movement and viral titers of CymMV in infected Phalaenopsis flowers and N. benthamiana leaves. Taking together, both the VSR and protein function of P126 might be prerequisites for unilaterally enhancing CymMV cell-to-cell movement by ORSV.