Transient expression of cauliflower mosaic virus (CaMV) P6-GFP complements a defective CaMV replicon to facilitate viral gene expression, replication and virion formation.

Over the past decades, several studies have examined the subcellular localization of the cauliflower mosaic virus (CaMV) P6 protein by tagging it with GFP (P6-GFP). These investigations have been essential in the development of models for inclusion body formation, nuclear transport, and microfilament-associated intracellular movement of P6 inclusion bodies for delivery of virions to plasmodesmata. Although it was shown early on that the translational transactivation function of P6-GFP was comparable to wild type P6, it has not been possible to incorporate a P6-GFP gene into an infectious clone of CaMV. Consequently, it has not been possible to formally prove that a P6-GFP fusion is comparable in function to the unmodified P6 protein. Here we show that transient expression of P6-GFP can complement a defective CaMV replicon through gene expression, replication and encapsidation, which validates the relevance of P6-GFP subcellular localization studies for understanding the development of CaMV infections.

Molecular characterization of viruses associated to leaf curl disease complex on zucchini squash in Iraq reveals Deng primer set could distinguish between New and Old World Begomoviruses.

The emergence of begomoviruses has led to significant losses in vegetable production in the recent years. Squash leaf curl disease (SqLCD) is caused by begomoviruses, and the infected plants show leaf curl symptoms on zucchini squash. In this study, we characterized the begomoviruses responsible for SqLCD symptoms and economic losses in zucchini major growing area near Baghdad and Babylon provinces. PCR amplification was performed to screen for begomovirus infection using Deng (a begomovirus specific) primer set. Sequence comparison confirmed the detection of two begomoviruses; Tomato leaf curl Palampur virus (ToLCPMV) and Squash leaf curl virus (SLCuV), in symptomatic zucchini samples when shared 99.14 and 99.50% maximum nucleotide (nt) identities with coat protein CP gene, respectively. All samples collected from Baghdad/Al-Jadriya were ToLCPMV infected while those collected from Babylon/Jibela were SLCuV infected. Moreover, mixed infection of the two viruses was detected in all zucchini squash samples collected from Baghdad/Yusufiyah. Evidence is provided here of the relevance of the wild species Malva neglecta and Datura stramonium as reservoir of begomoviruses that cause epedemics of leaf curl disease in zucchini squash in Iraq. Neighbor joining (NJ) Phylogenetic analysis confirmed the relatedness when diverged virus sequences in separated groups based on CP gene. The high nt identity suggests the two begomoviruses may recently be introduced to Iraq and could be a serious threatening to squash cultivation.

Tracing the Lineage of Two Traits Associated with the Coat Protein of the Tombusviridae: Silencing Suppression and HR Elicitation in Nicotiana Species.

In this paper we have characterized the lineage of two traits associated with the coat proteins (CPs) of the tombusvirids: Silencing suppression and HR elicitation in Nicotiana species. We considered that the tombusvirid CPs might collectively be considered an effector, with the CP of each CP-encoding species comprising a structural variant within the family. Thus, a phylogenetic analysis of the CP could provide insight into the evolution of a pathogen effector. The phylogeny of the CP of tombusvirids indicated that CP representatives of the family could be divided into four clades. In two separate clades the CP triggered a hypersensitive response (HR) in Nicotiana species of section Alatae but did not have silencing suppressor activity. In a third clade the CP had a silencing suppressor activity but did not have the capacity to trigger HR in Nicotiana species. In the fourth clade, the CP did not carry either function. Our analysis illustrates how structural changes that likely occurred in the CP effector of progenitors of the current genera led to either silencing suppressor activity, HR elicitation in select Nicotiana species, or neither trait.

A Novel Assay Based on Confocal Microscopy to Test for Pathogen Silencing Suppressor Functions.

In plants, RNA silencing is an important mechanism for gene regulation and defense that is targeted by proteins of viral pathogens effecting silencing suppression. In this chapter we describe a new assay to probe silencing suppressor activity using Agrobacterium infiltration of Nicotiana benthamiana and confocal microscopy. The key element in this assay involves the use of a reporter construct that is transiently expressed at a much lower level than free GFP, and this increases the sensitivity of detection of weak silencing suppressors such as the P6 protein of Cauliflower mosaic virus. Although initially developed for virus silencing suppressors, this technique could also prove valuable to characterize the potential for weak silencing suppressors in the effector repertoires of fungi, bacteria, nematodes, and oomycetes.

The P1 gene of Cauliflower mosaic virus is responsible for breaking resistance in Arabidopsis thaliana ecotype Enkheim (En-2).

Arabidopsis thaliana ecotype En-2 is resistant to several strains of Cauliflower mosaic virus (CaMV), including strain W260, but is susceptible to strain NY8153. Resistance in En-2 is conditioned by a single, semi-dominant gene called CAR1. We constructed several recombinant infectious clones between W260 and NY8153 and evaluated their capability to infect En-2. This analysis showed that the capacity of NY8153 to break resistance in En-2 was conditioned by mutations within the CaMV gene 1, a gene that encodes a protein dedicated to cell-to-cell movement (P1), and conversely, that P1 of W260 is responsible for eliciting the plant defense response. A previous study had shown that P6 of W260 was responsible for overcoming resistance in Arabidopsis ecotype Tsu-0 and that P6 of CaMV strain CM1841 was responsible for triggering resistance. The present study now shows that a second gene of CaMV is targeted by Arabidopsis for plant immunity.