Transcriptomic and Metabolomic Investigation on Leaf Necrosis Induced by ZmWus2 Transient Overexpression in Nicotiana benthamiana.

WUSCHEL (WUS) is a crucial transcription factor in regulating plant stem cell development, and its expression can also improve genetic transformation. However, the ectopic expression of WUS always causes pleiotropic effects during genetic transformation, making it important to understand the regulatory mechanisms underlying these phenomena. In our study, we found that the transient expression of the maize WUS ortholog ZmWus2 caused severe leaf necrosis in Nicotiana benthamiana. We performed transcriptomic and non-target metabolomic analyses on tobacco leaves during healthy to wilted states after ZmWus2 transient overexpression. Transcriptomic analysis revealed that ZmWus2 transformation caused active metabolism of inositol trisphosphate and glycerol-3-phosphate, while also upregulating plant hormone signaling and downregulating photosystem and protein folding pathways. Metabolomic analysis mainly identified changes in the synthesis of phenylpropanoid compounds and various lipid classes, including steroid synthesis. In addition, transcription factors such as ethylene-responsive factors (ERFs), the basic helix-loop-helix (bHLH) factors, and MYBs were found to be regulated by ZmWus2. By integrating these findings, we developed a WUS regulatory model that includes plant hormone accumulation, stress responses, lipid remodeling, and leaf necrosis. Our study sheds light on the mechanisms underlying WUS ectopic expression causing leaf necrosis and may inform the development of future genetic transformation strategies.

The plant protein NbP3IP directs degradation of Rice stripe virus p3 silencing suppressor protein to limit virus infection through interaction with the autophagy-related protein NbATG8.

In plants, autophagy is involved in responses to viral infection. However, the role of host factors in mediating autophagy to suppress viruses is poorly understood. A previously uncharacterized plant protein, NbP3IP, was shown to interact with p3, an RNA-silencing suppressor protein encoded by Rice stripe virus (RSV), a negative-strand RNA virus. The potential roles of NbP3IP in RSV infection were examined. NbP3IP degraded p3 through the autophagy pathway, thereby affecting the silencing suppression activity of p3. Transgenic overexpression of NbP3IP conferred resistance to RSV infection in Nicotiana benthamiana. RSV infection was promoted in ATG5- or ATG7-silenced plants and was inhibited in GAPC-silenced plants where autophagy was activated, confirming the role of autophagy in suppressing RSV infection. NbP3IP interacted with NbATG8f, indicating a potential selective autophagosomal cargo receptor role for P3IP. Additionally, the rice NbP3IP homolog (OsP3IP) also mediated p3 degradation and interacted with OsATG8b and p3. Through identification of the involvement of P3IP in the autophagy-mediated degradation of RSV p3, we reveal a new mechanism to antagonize the infection of RSV, and thereby provide the first evidence that autophagy can play an antiviral role against negative-strand RNA viruses.

Characterization of Proteins Involved in Chloroplast Targeting Disturbed by Rice Stripe Virus by Novel Protoplast⁻Chloroplast Proteomics.

Rice stripe virus (RSV) is one of the most devastating viral pathogens in rice and can also cause the general chlorosis symptom in Nicotiana benthamiana plants. The chloroplast changes associated with chlorosis symptom suggest that RSV interrupts normal chloroplast functions. Although the change of proteins of the whole cell or inside the chloroplast in response to RSV infection have been revealed by proteomics, the mechanisms resulted in chloroplast-related symptoms and the crucial factors remain to be elucidated. RSV infection caused the malformation of chloroplast structure and a global reduction of chloroplast membrane protein complexes in N. benthamiana plants. Here, both the protoplast proteome and the chloroplast proteome were acquired simultaneously upon RSV infection, and the proteins in each fraction were analyzed. In the protoplasts, 1128 proteins were identified, among which 494 proteins presented significant changes during RSV; meanwhile, 659 proteins were identified from the chloroplasts, and 279 of these chloroplast proteins presented significant change. According to the label-free LC⁻MS/MS data, 66 nucleus-encoded chloroplast-related proteins (ChRPs), which only reduced in chloroplast but not in the whole protoplast, were identified, indicating that these nuclear-encoded ChRPswere not transported to chloroplasts during RSV infection. Gene ontology (GO) enrichment analysis confirmed that RSV infection changed the biological process of protein targeting to chloroplast, where 3 crucial ChRPs (K4CSN4, K4CR23, and K4BXN9) were involved in the regulation of protein targeting into chloroplast. In addition to these 3 proteins, 41 among the 63 candidate proteins were characterized to have chloroplast transit peptides. These results indicated that RSV infection changed the biological process of protein targeting into chloroplast and the location of ChRPs through crucial protein factors, which illuminated a new layer of RSV⁻host interaction that might contribute to the symptom development.

p15 encoded by Garlic virus X is a pathogenicity factor and RNA silencing suppressor.

Garlic virus X (GarVX) encodes a 15 kDa cysteine-rich protein (CRP). To investigate the function(s) of p15, its subcellular localization, role as a symptom determinant and capacity to act as a viral suppressor of RNA silencing (VSR) were analysed. Results showed that GFP-tagged p15 was distributed in the cytoplasm, nucleus and nucleolus. Expression of p15 from PVX caused additional systemic foliar malformation and led to increased accumulation of PVX, showing that p15 is a virulence factor for reconstructed PVX-p15. Moreover, using a transient agro-infiltration patch assay and a Turnip crinkle virus (TCV) movement complementation assay, it was demonstrated that p15 possesses weak RNA silencing suppressor activity. Removal of an amino acid motif resembling a nuclear localization signal (NLS) prevented p15 from accumulating in the nucleus but did not abolish its silencing suppression activity. This study provides the first insights into the multiple functions of the GarVX p15 protein.

Complete genomic characterization of milk vetch dwarf virus isolates from cowpea and broad bean in Anhui province, China.

Cowpea and broad bean plants showing severe stunting and leaf rolling symptoms were observed in Hefei city, Anhui province, China, in 2014. Symptomatic plants from both species were shown to be infected with milk vetch dwarf virus (MDV) by PCR. The complete genomes of MDV isolates from cowpea and broad bean were sequenced. Each of them had eight genomic DNAs that differed between the two isolates by 10.7% in their overall nucleotide sequences. In addition, the MDV genomes from cowpea and broad bean were associated with two and three alphasatellite DNAs, respectively. This is the first report of MDV on cowpea in China and the first complete genome sequences of Chinese MDV isolates.

Functional identification of two minor capsid proteins from Chinese wheat mosaic virus using its infectious full-length cDNA clones.

Full-length cDNA clones of Chinese wheat mosaic virus (CWMV) RNA1 and RNA2 were produced from single reverse transcription PCR reactions and transcripts were shown to be infectious in both wheat and Nicotiana benthamiana. An efficient and reliable agro-infiltration method was then developed for reverse genetic assays in N. benthamiana. Inoculation of infectious cDNA clones resulted in obvious chlorotic symptoms, and CWMV viral genomic RNAs, capsid protein (CP)-related proteins, and typical rod-shaped particles were detectable on the inoculated and upper leaves, similar to those of WT virus. The optimal temperature for virus multiplication was 12 °C, but the optimum for systematic infection in plants was 17 °C. Mutant clones that abolished the N- or C-terminal extensions of the major CP did not inhibit systemic infection or the formation of rod-shaped particles but sometimes modified the symptoms in inoculated plants. These results suggest that the two minor CP-related proteins of CWMV are dispensable for viral infection, replication, systemic movement and virion assembly in plants.

P5-2 of rice black-streaked dwarf virus is a non-structural protein targeted to chloroplasts.

The genome segment S5 of rice black-streaked dwarf virus (genus Fijivirus, family Reoviridae) is functionally bicistronic in infected plants. It has a conserved second ORF (P5-2) partially overlapping the major ORF in a different reading frame, but its function remains unknown. P5-2 was detected in infected plants, but not in purified viral particles by Western blotting, indicating that it is a non-structural protein. In immunoelectron microscopy, polyclonal antibodies against P5-2 specifically labelled chloroplasts of infected rice plants. When P5-2 fused with green fluorescent protein was transiently expressed in leaves of Nicotiana benthamiana, fluorescence was also co-localized with chloroplasts. Experiments with deletion mutants of P5-2 showed that its N-terminal part was responsible for its targeting to chloroplasts.

Uptake, accumulation and toxicity of short chain chlorinated paraffins to wheat (Triticum aestivum L.).

Short chain chlorinated paraffins (SCCPs) are ubiquitous persistent organic pollutants. They have been widely detected in plant-based foods and might cause adverse impacts on humans. Nevertheless, uptake and accumulation mechanisms of SCCPs in plants remain unclear. In this study, the soil culture data indicated that SCCPs were strongly absorbed by roots (root concentration factor, RCF>1) yet limited translocated to shoots (translocation factor<1). The uptake mechanism was explored by hydroponic exposure, showing that hydrophobicity and molecular size influenced the root uptake and translocation of SCCPs. RCFs were significantly correlated with logKow values and molecular weights in a parabolic curve relationship. Besides, it was extremely difficult for SCCPs to translocate from shoots back to roots via phloem. An active energy-dependent process was proposed to be involved in the root uptake of SCCPs, which was supported by the uptake inhibition by the low temperature and metabolic inhibitor. Though SCCPs at environmentally relevant concentrations had no negative impacts on root morphology and chlorophyll contents, it caused obvious changes in cellular ultrastructure of root tip cells and induced a significant increase in superoxide dismutase activity. This information may be beneficial to moderate crop contamination by SCCPs, and to remedy soils polluted by SCCPs with plants.

Expression quantitative trait locus studies in the era of single-cell omics.

Genome-wide association studies have revealed that the regulation of gene expression bridges genetic variants and complex phenotypes. Profiling of the bulk transcriptome coupled with linkage analysis (expression quantitative trait locus (eQTL) mapping) has advanced our understanding of the relationship between genetic variants and gene regulation in the context of complex phenotypes. However, bulk transcriptomics has inherited limitations as the regulation of gene expression tends to be cell-type-specific. The advent of single-cell RNA-seq technology now enables the identification of the cell-type-specific regulation of gene expression through a single-cell eQTL (sc-eQTL). In this review, we first provide an overview of sc-eQTL studies, including data processing and the mapping procedure of the sc-eQTL. We then discuss the benefits and limitations of sc-eQTL analyses. Finally, we present an overview of the current and future applications of sc-eQTL discoveries.

Unveiling the spatial distribution and molecular mechanisms of terpenoid biosynthesis in Salvia miltiorrhiza and S. grandifolia using multi-omics and DESI-MSI.

Salvia miltiorrhiza and S. grandifolia are rich in diterpenoids and have therapeutic effects on cardiovascular diseases. In this study, the spatial distribution of diterpenoids in both species was analyzed by a combination of metabolomics and mass spectrometry imaging techniques. The results indicated that diterpenoids in S. miltiorrhiza were mainly abietane-type norditerpenoid quinones with a furan or dihydrofuran D-ring and were mainly distributed in the periderm of the roots, e.g. cryptotanshinone and tanshinone IIA. The compounds in S. grandifolia were mainly phenolic abietane-type tricyclic diterpenoids with six- or seven-membered C-rings, and were widely distributed in the periderm, phloem, and xylem of the roots, e.g. 11-hydroxy-sugiol, 11,20-dihydroxy-sugiol, and 11,20-dihydroxy-ferruginol. In addition, the leaves of S. grandifolia were rich in tanshinone biosynthesis precursors, such as 11-hydroxy-sugiol, while those of S. miltiorrhiza were rich in phenolic acids. Genes in the upstream pathway of tanshinone biosynthesis were highly expressed in the root of S. grandifolia, and genes in the downstream pathway were highly expressed in the root of S. miltiorrhiza. Here, we describe the specific tissue distributions and mechanisms of diterpenoids in two Salvia species, which will facilitate further investigations of the biosynthesis of diterpenoids in plant synthetic biology.

Identification of viral particles in the apoplast of Nicotiana benthamiana leaves infected by potato virus X.

The apoplast is the extracellular space for signalling, nutrient transport, and plant-microbe interactions, but little is known about how plant viruses use the foliar apoplast. Proteomic analysis of the apoplasts isolated from potato virus X (PVX)-infected Nicotiana benthamiana plants showed that the coat protein (CP) is the dominant viral component. The presence of the CP in the apoplast was confirmed by western blot, viral nucleic acid was detected by reverse transcription-PCR and northern blot, and viral particles were observed by transmission electron microscopy (TEM). The apoplast from infected leaves was infectious if rubbed onto healthy leaves but not when infiltrated into them. The exosomes were separated from the apoplast fluid by high-speed centrifugation and TEM showed that PVX particles were not associated with the exosomes. These results suggest that PVX virions are released to the N. benthamiana apoplast in a one-way manner and do not share the bidirectional transport of exosomes.

Endoplasmic reticulum remodeling induced by Wheat yellow mosaic virus infection studied by transmission electron microscopy.

Plant virus was a kind of organism lived depending on infecting viable host cell and propagated their posterity by replicating its hereditary nucleotide, transcripting into protein, assembling protein and nucleotide into virion (Ortín and Parra, 2006; Sanfaçon, 2005). Viral infection usually induces remodeling of host cell, especially endoplasmic reticulum (ER) for generating membrane packed viral factory. During the infection of Bymovirus, a kind of membranous body (MB) was generated in host cells, which is thought as an ER aggregate. In present study we performed a study on Wheat yellow mosaic virus (WYMV) induced MB by several transmission electron microscopy (TEM) based methods, including cytological observation, component analysis by immuno-gold labeling and structural analysis by electron tomography (ET). WYMV infection induced at least two morphologies of MB, including the lamella dominated morphology (lamella-MB) looked like sprawling cirrus, and the tubule dominated morphology (tubule-MB) looked like latticed network. MB was verified composing of ER as revealed by immuno-gold labeling by antibody against endoplasmic reticulum (ER) retention signal as well as by detailed observation of MB construction modules as double layer membrane. By immuno-gold labeling, both two MB morphologies (lamella-MB and tubule-MB) had same components in viral derived protein and membrane origination (from ER). Structural analysis by ET reconstruction revealed the organization of ER in MB. Lamella-MB was composed of cesER like structures arranged irregularly whereas tubule-MB was composed of tubER like structures arranged regularly. This study provided insights into the structural details in how Bymovirus utilizing host membrane system.

Phloem-limited reoviruses universally induce sieve element hyperplasia and more flexible gateways, providing more channels for their movement in plants.

Virion distribution and ultrastructural changes induced by the infection of maize or rice with four different reoviruses were examined. Rice black streaked dwarf virus (RBSDV, genus Fijivirus), Rice ragged stunt virus (RRSV, genus Oryzavirus), and Rice gall dwarf virus (RGDV, genus Phytoreovirus) were all phloem-limited and caused cellular hyperplasia in the phloem resulting in tumors or vein swelling and modifying the cellular arrangement of sieve elements (SEs). In contrast, virions of Rice dwarf virus (RDV, genus Phytoreovirus) were observed in both phloem and mesophyll and the virus did not cause hyperplasia of SEs. The three phloem-limited reoviruses (but not RDV) all induced more flexible gateways at the SE-SE interfaces, especially the non-sieve plate interfaces. These flexible gateways were also observed for the first time at the cellular interfaces between SE and phloem parenchyma (PP). In plants infected with any of the reoviruses, virus-like particles could be seen within the flexible gateways, suggesting that these gateways may serve as channels for the movement of plant reoviruses with their large virions between SEs or between SEs and PP. SE hyperplasia and the increase in flexible gateways may be a universal strategy for the movement of phloem-limited reoviruses.

Structure and components of the globular and filamentous viroplasms induced by Rice black-streaked dwarf virus.

Viroplasms of members of the family Reoviridae are considered to be viral factories for genome replication and virion assembly. Globular and filamentous phenotypes have different components and probably have different functions. We used transmission electron microscopy and electron tomography to examine the structure and components of the two viroplasm phenotypes induced by Rice black-streaked dwarf virus (RBSDV). Immuno-gold labeling was used to localize each of the 13 RBSDV encoded proteins as well as double-stranded RNA, host cytoskeleton actin-11 and α-tubulin. Ten of the RBSDV proteins were localized in one or both types of viroplasm. P5-1, P6 and P9-1 were localized on both viroplasm phenotypes but P5-1 was preferentially associated with filaments and P9-1 with the matrix. Structural analysis by electron tomography showed that osmiophilic granules 6-8nm in diameter served as the fundamental unit for constructing both of the viroplasm phenotypes but were more densely packed in the filamentous phenotype.

Characterization and Comparative Expression Profiling of Browning Response in Medinilla formosana after Cutting.

Plant browning is a recalcitrant problem for in vitro culture and often leads to poor growth of explants and even failure of tissue culture. However, the molecular mechanisms underlying browning-induced physiological processes remain unclear. Medinilla is considered one of the most difficult genera for tissue culture owning to its severe browning. In the present study, intact aseptic plantlets of Medinilla formosana Hayata previously obtained by ovary culture, were used to explore the characteristics and molecular mechanism of the browning response. Successive morphological and anatomical observations after cutting showed that the browning of M. formosana was not lethal but adaptive. De novo transcriptome and digital gene expression (DGE) profiling using Illumina high-throughput sequencing were then used to explore molecular regulation after cutting. About 7.5 million tags of de novo transcriptome were obtained and 58,073 unigenes were assembled and annotated. A total of 6,431 differentially expressed genes (DEGs) at three stages after cutting were identified, and the expression patterns of these browning-related genes were clustered and analyzed. A number of putative DEGs involved in signal transduction and secondary metabolism were particularly studied and the potential roles of these cutting-responsive mRNAs in plant defense to diverse abiotic stresses are discussed. The DGE profiling data were also validated by quantitative RT-PCR analysis. The data obtained in this study provide an excellent resource for unraveling the molecular mechanisms of browning processes during in vitro tissue culture, and lay a foundation for future studies to inhibit and eliminate browning damage.