Differential symptom development and viral RNA loads in ten Nicotiana benthamiana accessions infected with the tobamovirus yellow tailflower mild mottle virus

Yellow tailflower mild mottle virus (YTMMV, genus Tobamovirus) was identified from wild plants of solanaceous species in Australia. Nicotiana benthamiana is a species indigenous to the arid north of Australia. N. benthamiana accession RA-4 or the ‘lab’ type, which has a mutant, functionally-defective, RNA-dependent RNA polymerase 1 (Rdr1) gene (Nb-Rdr1m), has played a significant role in plant virology, but little study has been done on responses to virus infection by other accessions of N. benthamiana. All wild-collected N. benthamiana accessions used in this study harboured wild-type Rdr1 genes (Nb-Rdr1). We compared symptoms of YTMMV infection and viral RNA load on RA-4 and nine wild-collected accessions of N. benthamiana from mainland Western Australia, an island, and the Northern Territory. After inoculation with YTMMV, RA-4 plants responded with systemic hypersensitivity and all individuals were dead 35 days post-inoculation (dpi). Plants of wild-collected accessions exhibited a range of symptoms, from mild to severe, and some, but not all, individuals died in the same period. Quantitative reverse transcription PCR revealed that the Rdr1 mutation was not a predictor of viral RNA load or symptom severity. For example, wild-collected A019412 plants carried over twice the viral RNA load of RA-4 plants, but symptom expression was moderate. For plants of most accessions, viral RNA load did not increase after 10 dpi. The exception was plants of accession Barrow-1, where viral RNA load was low until 15 dpi, after which it increased over 29-fold. This study revealed differential responses by N. benthamiana accessions to infection by an isolate of YTMMV. The Rdr1 gene, whether mutant or wild-type, did not appear to influence viral RNA load or disease expression. Genetic diversity of the ten N. benthamiana lines in some cases reflected geographical location, but in other lines this was not so.

Systemic Sclerosis

Systemic sclerosis (SSc) is a chronic, autoimmune disease which can affect the blood vessels, the visceral organs, and the skin. SSc, most commonly, develops between the ages of 30 and 50, but it can be seen at any age. In terms of skin involvement, SSc can be classified as limited or diffuse. Its etiopathogenesis is still unclear. Microvascular dysfunction is thought to be followed by immunological activation, collagen and extracellular matrix deposition, and finally fibrosis. Diagnosis is based on clinical presentation. Sclerosis of the metacarpophalangeal and/or metatarsophalangeal joints is the major diagnostic criterion, whereas sclerodactylia, digital ulcers (DU), and pulmonary fibrosis are the minor criteria. SSc is diagnosed with one major criterion or two minor criteria. Detection of autoantibodies can help the diagnosis. Antinuclear antibody (ANA), anti-centromere antibody, anti-scl 70, RNA polymerase 1 and 3, and anti-fibrillin antibody can be found positive in SSc. SSc must be differentiated from all sclerosing diseases and the diseases with Raynaud’s phenomenon. Visceral diseases, such as primary pulmonary hypertension, primary biliary cirrhosis, and infiltrative cardiomyopathy, should also be considered in its differential diagnosis. The main treatment goal is to target visceral involvement.

Advances at the interface of cancer and systemic sclerosis

The interface between systemic sclerosis and cancer has offered valuable insights into our understanding of systemic sclerosis disease pathogenesis. Defining systemic sclerosis subgroups both temporally and serologically has been instrumental in stratifying cancer risk, with autoantibodies to RNA polymerase 3, RNA polymerase 1 large subunit, RNA binding region containing 3, and centromere identifying subgroups at increased or decreased risk of cancer. Clinically, improved subgrouping of systemic sclerosis patients provides the opportunity to detect cancer at earlier stages of disease while increasing our efficiency of cancer assessment. Additional studies are needed to define the optimal approach to cancer screening in systemic sclerosis, and validation studies in different cohorts will be needed to confirm all findings.

Global Transcriptomic Analysis Reveals Insights into the Response of ‘Etrog’ Citron (Citrus medica L.) to Citrus Exocortis Viroid Infection

Citrus exocortis viroid (CEVd) is the causal agent of citrus exocortis disease. We employed CEVd-infected ‘Etrog’ citron as a system to study the feedback regulation mechanism using transcriptome analysis in this study. Three months after CEVd infection, the transcriptome of fresh leaves was analyzed, and 1530 differentially expressed genes were detected. The replication of CEVd in citron induced upregulation of genes encoding key proteins that were involved in the RNA silencing pathway such as Dicer-like 2, RNA-dependent RNA polymerase 1, argonaute 2, argonaute 7, and silencing defective 3, as well as those genes encoding proteins that are related to basic defense responses. Many genes involved in secondary metabolite biosynthesis and chitinase activity were upregulated, whereas other genes related to cell wall and phytohormone signal transduction were downregulated. Moreover, genes encoding disease resistance proteins, pathogenicity-related proteins, and heat shock cognate 70 kDa proteins were also upregulated in response to CEVd infection. These results suggest that basic defense and RNA silencing mechanisms are activated by CEVd infection, and this information improves our understanding of the pathogenesis of viroids in woody plants.

Plant A20/AN1 proteins coordinate different immune responses including RNAi pathway for antiviral immunity

ABSTRACTSalicylic acid (SA)-mediated immunity plays important roles in combating virus in plants. Two plant stress associated protein (SAPs) containing dual A20/AN1 zinc-finger domain were found to play important roles in SA-mediated immunity; however, detailed mechanisms remain elusive. In this study, another orchid homolog gene of Pha13, Pha21, was analyzed. Pha21 confers antiviral immunity in both transgenic orchid and Arabidopsis overexpressing Pha21. Expression of Pha21 is early-induced by SA treatment, and is involved in the expression of the orchid homolog of the master regulator NPR1. Pha21 but not Pha13 is involved in the expression of key RNAi-related genes, Dicer-like nuclease 4 (DCL4) and Argonaut 1 (AGO1) in orchids. The involvement of SAPs in expression of orchid DCL4 and AGO1 is not limited to orchid, as AtSAP5 also plays essential role in the expression of Arabidopsis DCL4 and AGO1. However, unlike Pha13 and AtSAP5, Pha21 does not play positive role in the expression of orchid homolog gene of RNA-dependent RNA polymerase 1 (RdR1), an important gene in RNAi pathway. Pha21 can be found localized in the nucleus, and confers self-E3 ligase and ubiquitin binding activities. Functional domain analysis revealed that both A20 and AN1 domains of Pha21 are required for decreasing virus accumulation, and the AN1 domain plays a more important role in the expression of orchid DCL4. Collectively, our data suggests SA regulated SAPs play important roles in antiviral immunity and is involved in delicate regulation of key genes in RNAi-mediated pathway.IMPORTANCESalicylic acid (SA)-mediated antiviral immunity plays an important role to protect plants from virus infection; however, the detailed mechanisms remain elusive. We previously demonstrated that two plant A20/AN1 proteins, orchid Pha13 and Arabidopsis AtSAP5, function similarly and serve as an important hub to regulate SA-mediated antiviral immunity. In this study, we identified another orchid A20/AN1 protein, Pha21, which is involved in SA-mediated antiviral immunity. In contrast to Pha13 and AtSAP5, Pha21 plays minor negative roles in the expression of PhaRdR1 (orchid homolog of RNA-dependent RNA polymerase 1). However, Pha21 and AtSAP5, but not Pha13, are involved in the expression of important players in RNAi pathway, Dicer-like nuclease 4 (DCL4) and Argonaut 1 (AGO1), in orchid and Arabidopsis. Our data demonstrates that plant A20/AN1 proteins are conserved players in SA-mediated antiviral resistance among plants, and provide links between the A20/AN1 proteins and the RNAi pathway.