Positive Selection in the Chloroplastic ATP-Synthase ß-Subunit and Its Relation to Virulence Factors.

The most paradigmatic examples of molecular evolution under positive selection involve genes related to the immune system. Recently, different chloroplastic factors have been shown to be important for plant defenses, among them, the α- and ß-subunits of the ATP synthase. The ß-subunit has been reported to interact with several viral proteins while both proteins have been implicated with sensitivity to tentoxin, a phytotoxin produced by the widespread fungus Alternaria alternata. Given the relation of both protein to virulence factors, we studied whether these proteins are evolving under positive selection. To this end, we used the dN/dS ratio to examine possible sites under positive selection in several Angiosperm clades. After examining 79 plant genera and 1232 species, we found three times more sites under pervasive diversifying selection in the N-terminal region of the ß-subunit compared to the α-subunit, supporting previous results which identified this region as responsible for interacting with viral proteins. Moreover, we found the site 83 of ß-subunit under positive selection in several plant genera, a site clearly related to the sensitivity to tentoxin according to biochemistry assays, which possibly reflects the selective pressure of the non-host specific tentoxin across various Angiosperm clades.

Plant Immune System Activation Upon Citrus Leprosis Virus C Infection Is Mimicked by the Ectopic Expression of the P61 Viral Protein.

Citrus leprosis virus C (CiLV-C, genus Cilevirus, family Kitaviridae) is an atypical virus that does not spread systemically in its plant hosts. Upon its inoculation by Brevipalpus mites, only localized lesions occur, and the infection remains limited to cells around mite feeding sites. Here, we aimed to gain insights into the putative causes of viral unfitness in plants by expanding the limited knowledge of the molecular mechanisms underlying plant/kitavirid interactions. Firstly, we quantified the CiLV-C viral RNAs during the infection in Arabidopsis thaliana plants using RT-qPCR and systematized it by defining three stages of distinguishing subgenomic and genomic RNA accumulation: i) 0-24 h after infestation, ii) 2-4 days after infestation (dai), and iii) 6-10 dai. Accordingly, the global plant response to CiLV-C infection was assessed by RNA-Seq at each period. Results indicated a progressive reprogramming of the plant transcriptome in parallel to the increasing viral loads. Gene ontology enrichment analysis revealed the induction of cell growth-related processes at the early stages of the infection and the triggering of the SA-mediated pathway, ROS burst and hypersensitive response (HR) at the presymptomatic stage. Conversely, infected plants downregulated JA/ET-mediated pathways and processes involved in the primary metabolism including photosynthesis. Marker genes of unfolded protein response were also induced, suggesting a contribution of the endoplasmic reticulum stress to the cell death caused by the viral infection. Finally, we transiently expressed CiLV-C proteins in Nicotiana benthamiana plants to undertake their roles in the elicited plant responses. Expression of the CiLV-C P61 protein consistently triggered ROS burst, upregulated SA- and HR-related genes, increased SA levels, reduced JA levels, and caused cell death. Mimicry of responses typically observed during CiLV-C-plant interaction indicates P61 as a putative viral effector causing the HR-like symptoms associated with the infection. Our data strengthen the hypothesis that symptoms of CiLV-C infection might be the outcome of a hypersensitive-like response during an incompatible interaction. Consequently, the locally restricted infection of CiLV-C, commonly observed across infections by kitavirids, supports the thesis that these viruses, likely arising from an ancestral arthropod-infecting virus, are unable to fully circumvent plant defenses.

Differential Accumulation of Innate- and Adaptive-Immune-Response-Derived Transcripts during Antagonism between Papaya Ringspot Virus and Papaya Mosaic Virus.

Papaya ringspot virus (PRSV), a common potyvirus infecting papaya plants worldwide, can lead to either antagonism or synergism in mixed infections with Papaya mosaic virus (PapMV), a potexvirus. These two unrelated viruses produce antagonism or synergism depending on their order of infection in the plant. When PRSV is inoculated first or at the same time as PapMV, the viral interaction is synergistic. However, an antagonistic response is observed when PapMV is inoculated before PRSV. In the antagonistic condition, PRSV is deterred from the plant and its drastic effects are overcome. Here, we examine differences in gene expression by high-throughput RNA sequencing, focused on immune system pathways. We present the transcriptomic expression of single and mixed inoculations of PRSV and PapMV leading to synergism and antagonism. Upregulation of dominant and hormone-mediated resistance transcripts suggests that the innate immune system participates in synergism. In antagonism, in addition to innate immunity, upregulation of RNA interference-mediated resistance transcripts suggests that adaptive immunity is involved.

Chloroplast Proteome of Nicotiana benthamiana Infected by Tomato Blistering Mosaic Virus.

Tymovirus is a genus of plant pathogenic viruses that infects several dicotyledonous plants worldwide, causing serious diseases in economically important crops. The known cytopathic effect on the host cell organelles involves chloroplast membrane deformation and the induction of vesicles in its periphery. These vesicles are known to be the location where tymoviral genomic RNA replication occurs. Tomato blistering mosaic virus (ToBMV) is a tymovirus recently identified in tomato plants in Brazil, which is able to infect several other plants, including tobacco. In this work, we investigated the chloroplast proteomic profile of ToBMV-infected N. benthamiana using bidimensional electrophoresis (2-DE) and mass spectrometry, aiming to study the virus-host interaction related to the virus replication and infection. A total of approximately 200 spots were resolved, out of which 36 were differentially abundant. Differential spots were identified by mass spectrometry including photosynthesis-related and defense proteins. We identified proteins that may be targets of a direct interaction with viral proteins, such as ATP synthase ß subunit, RNA polymerase beta-subunit, 50S ribosomal protein L6 and Trigger factor-like protein. The identification of these candidate proteins gives support for future protein-protein interaction studies to confirm their roles in virus replication and disease development.

Transcriptome analysis provides insights into the delayed sticky disease symptoms in Carica papaya.

KEY MESSAGE: Global gene expression analysis indicates host stress responses, mainly those mediated by SA, associated to the tolerance to sticky disease symptoms at pre-flowering stage in Carica papaya. Carica papaya plants develop the papaya sticky disease (PSD) as a result of the combined infection of papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), or PMeV complex. PSD symptoms appear only after C. papaya flowers. To understand the mechanisms involved in this phenomenon, the global gene expression patterns of PMeV complex-infected C. papaya at pre-and post-flowering stages were assessed by RNA-Seq. The result was 633 and 88 differentially expressed genes at pre- and post-flowering stages, respectively. At pre-flowering stage, genes related to stress and transport were up-regulated while metabolism-related genes were down-regulated. It was observed that induction of several salicylic acid (SA)-activated genes, including PR1, PR2, PR5, WRKY transcription factors, ROS and callose genes, suggesting SA signaling involvement in the delayed symptoms. In fact, pre-flowering C. papaya treated with exogenous SA showed a tendency to decrease the PMeV and PMeV2 loads when compared to control plants. However, pre-flowering C. papaya also accumulated transcripts encoding a NPR1-inhibitor (NPR1-I/NIM1-I) candidate, genes coding for UDP-glucosyltransferases (UGTs) and several genes involved with ethylene pathway, known to be negative regulators of SA signaling. At post-flowering, when PSD symptoms appeared, the down-regulation of PR-1 encoding gene and the induction of BSMT1 and JA metabolism-related genes were observed. Hence, SA signaling likely operates at the pre-flowering stage of PMeV complex-infected C. papaya inhibiting the development of PSD symptoms, but the induction of its negative regulators prevents the full-scale and long-lasting tolerance.

Silencing of a Germin-Like Protein Gene (CchGLP) in Geminivirus-Resistant Pepper (Capsicum chinense Jacq.) BG-3821 Increases Susceptibility to Single and Mixed Infections by Geminiviruses PHYVV and PepGMV.

Germin-like proteins (GLPs) are encoded by a family of genes found in all plants, and in terms of function, the GLPs are implicated in the response of plants to biotic and abiotic stresses. CchGLP is a gene encoding a GLP identified in a geminivirus-resistant Capsicum chinense Jacq accession named BG-3821, and it is important in geminivirus resistance when transferred to susceptible tobacco in transgenic experiments. To characterize the role of this GLP in geminivirus resistance in the original accession from which this gene was identified, this work aimed at demonstrating the possible role of CchGLP in resistance to geminiviruses in Capsicum chinense Jacq. BG-3821. Virus-induced gene silencing studies using a geminiviral vector based in PHYVV component A, displaying that silencing of CchGLP in accession BG-3821, increased susceptibility to geminivirus single and mixed infections. These results suggested that CchGLP is an important factor for geminivirus resistance in C. chinense BG-3821 accession.

Identification of host proteins modulated by the virulence factor AC2 of Tomato chlorotic mottle virus in Nicotiana benthamiana.

Tomato, one of the most important crops cultivated worldwide, has been severely affected by begomoviruses such as the Tomato chlorotic mottle virus (ToCMoV). Virulence factor AC2 is considered crucial for a successful virus-plant interaction and is known to act as a transcriptional activator and in some begomoviruses to function as an RNA silencing suppressor factor. However, the exact functions of the AC2 protein of the begomovirus ToCMoV are not yet established. The aim of the present study was to identify differentially expressed proteins of the model plant Nicotiana benthamiana in response to the expression of the AC2 gene, isolated from ToCMoV. N. benthamiana plants were inoculated with Agrobacterium tumefaciens containing the viral vector Potato virus X (PVX) and with the PVX-AC2 construction. 2DE was performed and proteins were identified by MS. The results showed that the expression of ToCMoV AC2 alters the levels of several host proteins, which are important for normal plant development, causing an imbalance in cellular homeostasis. This study highlights the effect of AC2 in the modulation of plant defense processes by increasing the expression of several oxidative stress-related and pathogenesis-related proteins, as well as its role in modulating the proteome of the photosynthesis and energy production systems.