N6-Methyladenosine Regulatory Machinery is Involved In Viral Infection of Plum Pox Virus And Potato Virus Y In Nicotiana Benthamiana

Abstract N6-methyladenosine (m6A) is a post-transcriptional modification of biological mRNA and non-coding RNAs, which by regulating the mRNA stability and translation. It has been demonstrated that m6A methylation has a regulatory effect on human RNA virus replication. In this project, Plum pox virus (PPV) and Potato Y virus (PVY) were used to examine the m6A modification in Nicotiana benthamiana during natural infection. The results showed that the global level of m6A in both PVY and PPV infected plants were significantly decreased than non-infected plants. Particularly, the PPV and PVY infection could alter the m6A level of the host endogenous gene. This is suggesting that plant viruses may disrupt the balance of the m6A in plant. Meanwhile, we found that viral genome RNA can be targeted by m6A methylation. Two m6A-enrich regions in PPV genome RNA and four in PVY genome RNA were detected, which are located in the coding region of viruses. Based on the ALKB and METTL sequences in the transcriptome sequencing data of the virus-infected plant, we cloned 2 NbALKB genes and 2 NbMETTL genes in N. benthamiana . According to results of transient expression and VIGS assay, NbALKB gene appears slightly contributing PPV and PVY infection. NbMETTL gene showed certain inhibition effect in PPV infection, but not PVY. Therefore, our data suggested that m6A methylation in plant might be an anti-viral strategy in some plant viruses.

Evaluation of the Chilli veinal mottle virus CP gene expressing transgenic Nicotiana benthamiana plants for disease resistance against the virus

Vegetables are an important source of income and high-value crops for small farmers. Chilli (Capsicum spp.) is one of the most economically important vegetables of Pakistan and it is grown throughout the country. It is a rich source of nutrition especially vitamins A, B, C and E along with minerals as folic acid, manganese (Mn), potassium (K) and molybdenum (Mo). Chilli possesses seven times more amount of vitamin C than an orange. Vitamin A, C and beta-carotenoids are strong antioxidants to scavenge the free radicals. Chilli production is restricted due to various biotic factors. Among these viruses, Chilli veinal mottle virus (ChiVMV) is one of the most destructive and menacing agents that inflicts heavy and colossal losses that accounted for 50% yield loss both in quality and quantity. Pathogen-Derived Resistance (PDR) approach is considered one of the effective approaches to manage plant viruses. In this study, ChiVMV was characterized on a molecular level, the coat protein (CP) gene of the virus was stably transformed into Nicotiana benthamiana plants using Agrobacterium tumefaciens. The transgenic plants were challenged with the virus to evaluate the level of resistance of plants against the virus. It was observed that the plants expressing CP gene have partial resistance against the virus in terms of symptoms’ development and virus accumulation. Translation of this technique into elite chilli varieties will be resulted to mitigate the ChiVMV in the crop as well as an economic benefit to the farmers.

Functional characterization of tobacco (Nicotiana benthamiana) serotonin N-acetyltransferases (NbSNAT1 and NbSNAT2)

Nicotiana benthamiana (tobacco) is an important dicotyledonous model plant; however, no serotonin N-acetyltransferases (SNATs) have been characterized in tobacco. In this study, we identified, cloned, and characterized the enzyme kinetics of two SNAT genes from N. benthamiana, NbSNAT1 and NbSNAT2. The substrate affinity (Km) and maximum reaction rate (Vmax) for NbSNAT1 were 579 µM and 136 pkat/mg protein for serotonin, and 945 µM and 298 pkat/mg protein for 5-methoxytryptamine, respectively. Similarly, the Km and Vmax values for NbSNAT2 were 326 µM and 26 pkat/mg protein for serotonin, and 872 µM and 92 pkat/mg protein for 5-methoxytryptamine, respectively. Moreover, we found that NbSNAT1 and NbSNAT2 localized to chloroplasts, similar to SNAT proteins from other plant species. The activities of the NbSNAT proteins were not affected by melatonin feedback inhibition in vitro. Finally, transgenic tobacco plants overexpressing either NbSNAT1 or NbSNAT2 did not exhibit increased melatonin levels, possibly due to the expression of catabolic enzymes. Generating transgenic tobacco plants with downregulated NbSNAT expression would provide further insight into the functional role of melatonin in tobacco plants.

NSvc4 Encoded by Rice Stripe Virus Targets Host Chloroplasts to Suppress Chloroplast-Mediated Defense

Our previous research found that NSvc4, the movement protein of rice stripe virus (RSV), could localize to the actin filaments, endoplasmic reticulum, plasmodesmata, and chloroplast, but the roles of NSvc4 played in the chloroplast were opaque. Here, we confirm the accumulation of NSvc4 in the chloroplasts and the N-terminal 1–73 amino acids of NSvc4 are sufficient to localize to chloroplasts. We provide evidence to show that chloroplast-localized NSvc4 can impair the chloroplast-mediated immunity. Expressing NSvc4 in Nicotiana benthamiana leaves results in the decreased expression of defense-related genes NbPR1, NbPR2, and NbWRKY12 and the inhibition of chloroplast-derived ROS production. In addition, generation of an infectious clone of potato virus X (PVX) carrying NSvc4 facilitates PVX infection in N. benthamiana plants. Moreover, we identify two chloroplast-related host factors, named NbGAPDH-A and NbPsbQ1, both of which can interact with NSvc4. Knockdown of NbGAPDH-A or NbPsbQ1 can both promote RSV infection. Our results decipher a detailed function of NSvc4 in the chloroplast.

Systemic Identification and Functional Characterization of Common in Fungal Extracellular Membrane Proteins in Lasiodiplodia theobromae

Plant pathogenic fungi deploy secreted proteins into apoplastic space or intracellular lumen to promote successful infections during plant-pathogen interactions. In the present study, fourteen CFEM domain-containing proteins were systemically identified in Lasiodiplodia theobromae and eight of them were functionally characterized. All eight proteins were confirmed to be secreted into extracellular space by a yeast signal peptide trapping system. The transcriptional levels of most CFEM genes, except for LtCFEM2 and LtCFEM6, were significantly elevated during infection. In addition, almost all LtCFEM genes, apart from LtCFEM2, LtCFEM3, and LtCFEM6, were transcriptionally up-regulated at 35°C in contrast to that at 25°C and 30°C. As two elicitors, LtCFEM1 induced local yellowish phenotype and LtCFEM4 triggered cell death in Nicotiana benthamiana leaves. Furthermore, these proteins displayed distinct subcellular localizations when expressed transiently in N. benthamiana. Moreover, two genes, LtCFEM7 and LtCFEM8, were found to be spliced alternatively by RT-PCR and sequencing. Therefore, our data suggest that LtCFEM proteins play important roles in multiple aspects, including pathogenicity and plant immune response, which will enhance our understanding of the sophisticated pathogenic mechanisms of plant opportunistic pathogen L. theobromae.

Evaluation of Crossability between Nicotiana benthamiana and Nicotiana excelsior

Wild tobacco species in the Nicotiana section Suaveolentes are promising genetic resources to introduce their disease resistance to cultivated tobacco, Nicotiana tabacum. However, hybrid lethality is observed in hybrid seedlings from crosses between most Suaveolentes species and N. tabacum. In particular, N. benthamiana belonging to the section Suaveolentes produces only viable hybrids after crossing with N. tabacum. In the present study, crossability between N. benthamiana and N. excelsior (section Suaveolentes) was investigated to test the possible usefulness of N. benthamiana as the bridge parent to transfer desirable genes of N. excelsior to N. tabacum via bridge crossing. After reciprocal crosses using three accessions of N. benthamiana and N. excelsior each, several crossing barriers such as cross-incompatibility, seed abortion, and male and female hybrid sterility were observed. Although reciprocal hybrids between N. benthamiana and N. excelsior showed a high degree of chromosome pairing in meiosis, univalents and multivalents, as well as chromosome bridges and lagging chromosomes, were observed. These meiotic abnormalities were thought to cause hybrid sterility. The possible usefulness of reciprocal hybrids between N. benthamiana and N. excelsior is discussed.