Selection and Validation of Reference Genes for RT-qPCR Analysis of Gene Expression in Nicotiana benthamiana upon Single Infections by 11 Positive-Sense Single-Stranded RNA Viruses from Four Genera

Zhang, Ge; Zhang, Zhuo; Wan, Qionglian; Zhou, Huijie; Jiao, Mengting; Zheng, Hongying; Lu, Yuwen; Rao, Shaofei; Wu, Guanwei; Chen, Jianping; Yan, Fei; Peng, Jiejun; Wu, Jian

doi:10.3390/plants12040857

Cited by 8 publications

(6 citation statements)

References 49 publications

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“…To select reference genes, we used a list of stably expressed plant genes previously established on Arabidopsis thaliana infected by several viruses (Zhang et al, 2023) . Orthologous genes were identified in Beta vulgaris using the KEGG database and primers compatible with TaqMan and SYBR Green detection were designed using the IDT Primer Request tool available online (https://eu.idtdna.com/pages/tools/primerquest).…”

Section: Methodsmentioning

confidence: 99%

Interplay between a polerovirus and a closterovirus decreases aphid transmission of the polerovirus

Khechmar,

Chesnais,

Villeroy

et al. 2024

Preprint

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Multi-infection of plant by viruses is very common and can change drastically infection parameters such as virus accumulation, distribution and vector transmission. Sugar beet is an important crop that is frequently co-infected by the polerovirus beet chlorosis virus (BChV) and the closterovirus beet yellows virus (BYV), both vectored by the green peach aphid (Myzus persicae). These phloem-limited viruses are acquired while aphids ingest phloem sap from infected plants. Here we found that co-infection decreased transmission of BChV by ~50 %, but had no impact on BYV transmission. The drastic reduction of BChV transmission was not due to lower accumulation of BChV in co-infected plants, nor to reduced phloem sap ingestion by aphids from these plants. Using the SABER-FISH in situ hybridization technique on plants, we observed that 40 % of the infected phloem cells were co-infected and that co-infection caused redistribution of BYV in these cells. The BYV accumulation pattern changed from distinct intracellular spherical inclusions in mono-infected cells to a diffuse form in co-infected cells. There, BYV co-localized with BChV throughout the cytoplasm, indicative of virus-virus interactions. We propose that BYV-BChV interactions could restrict BChV access to the sieve tubes and reduce its accessibility for aphids and present a model of how co-infection could alter BChV intracellular movement and/or phloem loading and reduce BChV transmission.

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Section: Methodsmentioning

confidence: 99%

Interplay between a polerovirus and a closterovirus decreases aphid transmission of the polerovirus

Khechmar,

Chesnais,

Villeroy

et al. 2024

Preprint

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“…Amplification of the genomes was achieved using KOD FX Neo high-fidelity DNA polymerase (TOYOBO, Japan) with specific primers. For quantitative PCR (qPCR), AceQ qPCR SYBR Green PCR Master Mix kit (Vazyme, Nanjing, China) was utilized, with the specificity of each reaction confirmed through melting curve analysis [42]. RT-qPCR was carried out on a LightCycler480 instrument (Roche, Basel, Switzerland) with 384-well PCR plates.…”

Section: Rna Extraction Rt-pcr and Rt-qpcrmentioning

confidence: 99%

“…Three biological replicates were included for each experiment, and three technical replicates were set for each biological replicate. The thermal cycling conditions for RT-qPCR were 95 • C for 30 s, followed by 40 cycles of 15 s at 95 • C, 14 s at 55 • C, and 28 s at 72 • C. Melting curves were prepared with the following thermal conditions: 95 • C for 10 s, 60 • C for 30 s, and 95 • C for 15 s. Relative gene expression was analyzed using the 2 −∆∆CT method [42]. All primers utilized in this study are listed in Table S1.…”

Section: Rna Extraction Rt-pcr and Rt-qpcrmentioning

confidence: 99%

Unveiling the Role of SlRNC1 in Chloroplast Development and Global Gene Regulation in Tomato Plants

Nie,

Zhang,

Wang

et al. 2024

IJMS

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RNC1, a plant-specific gene, is known for its involvement in splicing group II introns within maize chloroplast. However, its role in chloroplast development and global gene expression remains poorly understood. This study aimed to investigate the role of RNC1 in chloroplast development and identify the genes that mediate its function in the development of entire tomato plants. Consistent with findings in maize, RNC1 silencing induced dwarfism and leaf whitening in tomato plants. Subcellular localization analysis revealed that the RNC1 protein is localized to both the nucleus and cytoplasm, including the stress granule and chloroplasts. Electron microscopic examination of tomato leaf transverse sections exposed significant disruptions in the spatial arrangement of the thylakoid network upon RNC1 silencing, crucial for efficient light energy capture and conversion into chemical energy. Transcriptome analysis suggested that RNC1 silencing potentially impacts tomato plant development through genes associated with all three categories (biological processes, cellular components, and molecular functions). Overall, our findings contribute to a better understanding of the critical role of RNC1 in chloroplast development and its significance in plant physiology.

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“…The RT-qPCR was performed using the CFX Connect detection system (Bio-Rad Laboratories, Mississauga, ON, Canada). ACT 1 and EF1α were used to normalize the transcript levels [51]. Each sample was run in triplicate and repeated six times from two pooled biological replicates of silenced and non-silenced plants.…”

Section: Quantitative Rt-pcrmentioning

confidence: 99%

Differential Participation of Plant Ribosomal Proteins from the Small Ribosomal Subunit in Protein Translation under Stress

2023

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Upon exposure to biotic and abiotic stress, plants have developed strategies to adapt to the challenges imposed by these unfavorable conditions. The energetically demanding translation process is one of the main elements regulated to reduce energy consumption and to selectively synthesize proteins involved in the establishment of an adequate response. Emerging data have shown that ribosomes remodel to adapt to stresses. In Arabidopsis thaliana, ribosomes consist of approximately eighty-one distinct ribosomal proteins (RPs), each of which is encoded by two to seven genes. Recent research has revealed that a mutation in a given single RP in plants can not only affect the functions of the RP itself but can also influence the properties of the ribosome, which could bring about changes in the translation to varying degrees. However, a pending question is whether some RPs enable ribosomes to preferentially translate specific mRNAs. To reveal the role of ribosomal proteins from the small subunit (RPS) in a specific translation, we developed a novel approach to visualize the effect of RPS silencing on the translation of a reporter mRNA (GFP) combined to the 5’UTR of different housekeeping and defense genes. The silencing of genes encoding for NbRPSaA, NbRPS5A, and NbRPS24A in Nicotiana benthamiana decreased the translation of defense genes. The NbRACK1A-silenced plant showed compromised translations of specific antioxidant enzymes. However, the translations of all tested genes were affected in NbRPS27D-silenced plants. These findings suggest that some RPS may be potentially involved in the control of protein translation.

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Selection and Validation of Reference Genes for RT-qPCR Analysis of Gene Expression in Nicotiana benthamiana upon Single Infections by 11 Positive-Sense Single-Stranded RNA Viruses from Four Genera

Cited by 8 publications

References 49 publications

Interplay between a polerovirus and a closterovirus decreases aphid transmission of the polerovirus

Interplay between a polerovirus and a closterovirus decreases aphid transmission of the polerovirus

Unveiling the Role of SlRNC1 in Chloroplast Development and Global Gene Regulation in Tomato Plants

Differential Participation of Plant Ribosomal Proteins from the Small Ribosomal Subunit in Protein Translation under Stress

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