The virulence factor p25 of beet necrotic yellow vein virus interacts with multiple Aux/IAA proteins from Beta vulgaris: Implications for rhizomania development

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is characterized by excessive lateral root (LR) formation. Auxin-mediated degradation of Aux/IAA transcriptional repressors stimulates gene regulatory networks leading to LR organogenesis and involves several Aux/IAA proteins acting at distinctive stages of LR development. Previously, we showed that BNYVV p25 virulence factor interacts with BvIAA28, a transcriptional repressor acting at early stages of LR initiation. The evidence suggested that p25 inhibits BvIAA28 nuclear localization, thus, de-repressing transcriptional network leading to LR initiation. However, it was not clear whether p25 interacts with other Aux/IAA proteins. Here, by adopting bioinformatics, in vitro and in vivo protein interaction approaches we show that p25 interacts also with BvIAA2 and BvIAA6. Moreover, we confirmed that the BNYVV infection is, indeed, accompanied by an elevated auxin level in the infected LRs. Nevertheless, expression levels of BvIAA2 and BvIAA6 remained unchanged upon BNYVV infection. Mutational analysis indicated that interaction of p25 with either BvIAA2 or BvIAA6 requires full-length proteins as even single amino acid residue substitutions abolished the interactions. Compared to p25-BvIAA28 interaction that leads to redistribution of BvIAA28 into cytoplasm, both BvIAA2 and BvIAA6 remained confined into the nucleus regardless of the presence of p25 suggesting their stabilization though p25 interaction. Overexpression of p25-interacting partners (BvIAA2, BvIAA6 and BvIAA28) in Nicotiana benthamiana induced an auxin-insensitive phenotype characterized by plant dwarfism and dramatically reduced LR development. Thus, our work reveals a distinct class of transcriptional repressors targeted by p25

Production of a Beet chlorosis virus full-length cDNA clone by means of Gibson assembly and analysis of biological properties

Beet chlorosis virus (genus Polerovirus, family Luteoviridae), which is persistently transmitted by the aphid Myzus persicae, is part of virus yellows in sugar beet and causes interveinal yellowing as well as significant yield loss in Beta vulgaris. To allow reverse genetic studies and replace vector transmission, an infectious cDNA clone under cauliflower mosaic virus 35S control in a binary vector for agrobacterium-mediated infection was constructed using Gibson assembly. Following agroinoculation, the BChV full-length clone was able to induce a systemic infection of the cultivated B. vulgaris. The engineered virus was successfully aphid-transmitted when acquired from infected B. vulgaris and displayed the same host plant spectrum as wild-type virus. This new polerovirus infectious clone is a valuable tool to identify the viral determinants involved in host range and study BChV protein function, and can be used to screen sugar beet for BChV resistance

The Virulence Factor p25 of Beet Necrotic Yellow Vein Virus Interacts With Multiple Aux/IAA Proteins From Beta vulgaris: Implications for Rhizomania Development

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is characterized by excessive lateral root (LR) formation. Auxin-mediated degradation of Aux/IAA transcriptional repressors stimulates gene regulatory networks leading to LR organogenesis and involves several Aux/IAA proteins acting at distinctive stages of LR development. Previously, we showed that BNYVV p25 virulence factor interacts with BvIAA28, a transcriptional repressor acting at early stages of LR initiation. The evidence suggested that p25 inhibits BvIAA28 nuclear localization, thus, de-repressing transcriptional network leading to LR initiation. However, it was not clear whether p25 interacts with other Aux/IAA proteins. Here, by adopting bioinformatics, in vitro and in vivo protein interaction approaches we show that p25 interacts also with BvIAA2 and BvIAA6. Moreover, we confirmed that the BNYVV infection is, indeed, accompanied by an elevated auxin level in the infected LRs. Nevertheless, expression levels of BvIAA2 and BvIAA6 remained unchanged upon BNYVV infection. Mutational analysis indicated that interaction of p25 with either BvIAA2 or BvIAA6 requires full-length proteins as even single amino acid residue substitutions abolished the interactions. Compared to p25-BvIAA28 interaction that leads to redistribution of BvIAA28 into cytoplasm, both BvIAA2 and BvIAA6 remained confined into the nucleus regardless of the presence of p25 suggesting their stabilization though p25 interaction. Overexpression of p25-interacting partners (BvIAA2, BvIAA6 and BvIAA28) in Nicotiana benthamiana induced an auxin-insensitive phenotype characterized by plant dwarfism and dramatically reduced LR development. Thus, our work reveals a distinct class of transcriptional repressors targeted by p25

Molecular, serological and biological variation among chickpea chlorotic stunt virus isolates from five countries of North Africa and West Asia

Chickpea chlorotic stunt virus (CpCSV), a proposed new member of the genus Polerovirus (family Luteoviridae), has been reported only from Ethiopia. In attempts to determine the geographical distribution and variability of CpCSV, a pair of degenerate primers derived from conserved domains of the luteovirus coat protein (CP) gene was used for RT-PCR analysis of various legume samples originating from five countries and containing unidentified luteoviruses. Sequencing of the amplicons provided evidence for the occurrence of CpCSV also in Egypt, Morocco, Sudan, and Syria. Phylogenetic analysis of the CP nucleotide sequences of 18 samples from the five countries revealed the existence of two geographic groups of CpCSV isolates differing in CP sequences by 8–10%. Group I included isolates from Ethiopia and Sudan, while group II comprised those from Egypt, Morocco and Syria. For distinguishing these two groups, a simple RFLP test using HindIII and/or PvuII for cleavage of CP-gene-derived PCR products was developed. In ELISA and immunoelectron microscopy, however, isolates from these two groups could not be distinguished with rabbit antisera raised against a group-I isolate from Ethiopia (CpCSV-Eth) and a group-II isolate from Syria (CpCSV-Sy). Since none of the ten monoclonal antibodies (MAbs) that had been produced earlier against CpCSV-Eth reacted with group-II isolates, further MAbs were produced. Of the seven MAbs raised against CpCSV-Sy, two reacted only with CpCSV-Sy and two others with both CpCSV-Sy and -Eth. This indicated that there are group I- and II-specific and common (species-specific) epitopes on the CpCSV CP and that the corresponding MAbs are suitable for specific detection and discrimination of CpCSV isolates. Moreover, CpCSV-Sy (group II) caused more severe stunting and yellowing in faba bean than CpCSV-Eth (group I). In conclusion, our data indicate the existence of a geographically associated variation in the molecular, serological and presumably biological properties of CpCSV

Two distinct nanovirus species infecting faba bean in Morocco

Using monoclonal antibodies raised against a Faba bean necrotic yellows virus (FBNYV) isolate from Egypt and a Faba bean necrotic stunt virus (FBNSV) isolate from Ethiopia, a striking serological variability among nanovirus isolates from faba bean in Morocco was revealed. To obtain a better understanding of this nanovirus variability in Morocco, the entire genomes of two serologically contrasting isolates referred to as Mor5 and Mor23 were sequenced. The eight circular ssDNA components, each identified from Mor5- and Mor23-infected tissues and thought to form the complete nanovirus genome, ranged in size from 952 to 1,005 nt for Mor5 and from 980 to 1,004 nt for Mor23 and were structurally similar to previously described nanovirus DNAs. However, Mor5 and Mor23 differed from each other in overall nucleotide and amino acid sequences by 25 and 26%, respectively. Mor23 was most closely related to typical FBNYV isolates described earlier from Egypt and Syria, with which it shared a mean amino acid sequence identity of about 94%. On the other hand, Mor5 most closely resembled a FBNSV isolate from Ethiopia, with which it shared a mean amino acid sequence identity of approximately 89%. The serological and genetic differences observed for Mor5 and Mor23 were comparable to those observed earlier for FBNYV, FBNSV, and Milk vetch dwarf virus. Following the guidelines on nanovirus species demarcation, this suggests that Mor23 and Mor5 represent isolates of FBNYV and FBNSV, respectively. This is the first report not only on the presence of FBNSV in a country other than Ethiopia but also on the occurrence and complete genome sequences of members of two nanovirus species in the same country, thus providing evidence for faba bean crops being infected by members of two distinct nanovirus species in a restricted geographic area

Molecular evolution of viral multifunctional proteins: the case of Potyvirus HC-Pro

[EN] Our knowledge on the mode of evolution of the multifunctional viral proteins remains incomplete. To tackle this problem, here, we have investigated the evolutionary dynamics of the potyvirus multifunctional protein HC-Pro, with particular focus on its functional domains. The protein was partitioned into the three previously described functional domains, and each domain was analyzed separately and assembled. We searched for signatures of adaptive evolution and evolutionary dependencies of amino acid sites within and between the three domains using the entire set of available potyvirus sequences in GenBank. Interestingly, we identified strongly significant patterns of co-occurrence of adaptive events along the phylogenetic tree in the three domains. These patterns suggest that Domain I, whose main function is to mediate aphid transmission, has likely been coevolving with the other two domains, which are involved in different functions but all requiring the capacity to bind RNA. By contrast, episodes of positive selection on Domains II and III did not correlate, reflecting a trade-off between their evolvability and their evolutionary dependency likely resulting from their functional overlap. Covariation analyses have identified several groups of amino acids with evidence of concerted variation within each domain, but interdomain significant covariations were only found for Domains II and III, further reflecting their functional overlappingThis work was supported by grants BFU2012-30805 (SFE) and BFU2012-36346 (MAF) from the Spanish Direccio´n General de Investigacio´n Cientı´fica y Te´cnica and by an EMBO Short-Term Fellowship and the Mentoring Program from the Foundation for Polish Science (BHJ).Hasiów-Jaroszewska, B.; Fares Riaño, MA.; Elena Fito, SF. (2014). Molecular evolution of viral multifunctional proteins: the case of Potyvirus HC-Pro. 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Molecular evidence for the occurrence of two new luteoviruses in cool season food legumes in Northeast Africa

Some legume samples with yellowing and stunting symptoms from Ethiopia and Sudan that serologically reacted with a broad-spectrum luteovirus monoclonal antibody did not react or veryweakly reacted with virus-specific antibodies suggesting the occurrence of new luteovirus variants. Reverse transcriptase (RT)-PCR amplification, cloning, nucleotide sequencing and analysis of coatprotein (CP) gene of a luteovirus isolate from chickpea in Sudan indicated that it shares a closest predicted amino acid sequence identity of only 66% with Soybean dwarf virus (SbDV). Since this is lessthan the accepted threshold value of 90% recommended for discriminating luteovirus species, the isolate is suggested to represent a distinct luteovirus for which the name Chickpea yellows virus(CpYV) is proposed. Similarly, a lentil isolate from Ethiopia shared a closest CP amino acid sequence identity of 86% with viruses of the Beet western yellows virus subgroup. Following the same criteria,this isolate represents another distinct luteovirus species for which the name Lentil stunt virus (LStV) is suggested. From faba bean, CP sequences of Turnip yellows virus were amplified from Egyptian andMoroccan samples whereas partial CP sequences of SbDV were amplified from Ethiopian, Syrian and Chinese samples. The study indicated that legume luteoviruses in northeast Africa are highly diverse

Molecular evidence for the occurrence of two new luteoviruses in cool season food legumes in Northeast Africa

Some legume samples with yellowing and stunting symptoms from Ethiopia and Sudan that serologically reacted with a broad-spectrum luteovirus monoclonal antibody did not react or very weakly reacted with virus-specific antibodies suggesting the occurrence of new luteovirus variants. Reverse transcriptase (RT)-PCR amplification, cloning, nucleotide sequencing and analysis of coat protein (CP) gene of a luteovirus isolate from chickpea in Sudan indicated that it shares a closest predicted amino acid sequence identity of only 66% with Soybean dwarf virus (SbDV). Since this is less than the accepted threshold value of 90% recommended for discriminating luteovirus species, the isolate is suggested to represent a distinct luteovirus for which the name Chickpea yellows virus (CpYV) is proposed. Similarly, a lentil isolate from Ethiopia shared a closest CP amino acid sequence identity of 86% with viruses of the Beet western yellows virus subgroup. Following the same criteria, this isolate represents another distinct luteovirus species for which the name Lentil stunt virus (LStV) is suggested. From faba bean, CP sequences of Turnip yellows virus were amplified from Egyptian and Moroccan samples whereas partial CP sequences of SbDV were amplified from Ethiopian, Syrian and Chinese samples. The study indicated that legume luteoviruses in northeast Africa are highly diverse