Interaction of Solanum tuberosum L. translation initiation factors eIF4E with potato virus Y VPg: Apprehend and avoid.

Potato virus Y (PVY) is one of the most dangerous agricultural pathogens that causes substantial harm to vegetative propagated crops, such as potatoes (Solanum tuberosum L.). A necessary condition for PVY infection is an interaction between the plant cap-binding translation initiation factors eIF4E and a viral protein VPg, which mimics the cap-structure. In this study, we identified the point mutations in potato eIF4E1 and eIF4E2 that disrupt VPg binding while preserving the functional activity. For the structural interpretation of the obtained results, molecular models of all the studied forms of eIF4E1 and eIF4E2 were constructed and analyzed via molecular dynamics. The results of molecular dynamics simulations corresponds to the biochemical results and suggests that the ß1ß2 loop plays a key role in the stabilization of both eIF4E-cap and eIF4E-VPg complexes.

Plant eIF4E isoforms as factors of susceptibility and resistance to potyviruses.

Potyviruses are the largest group of plant-infecting RNA viruses that affect a wide range of crop plants. Plant resistance genes against potyviruses are often recessive and encode translation initiation factors eIF4E. The inability of potyviruses to use plant eIF4E factors leads to the development of resistance through a loss-of-susceptibility mechanism. Plants have a small family of eIF4E genes that encode several isoforms with distinct but overlapping functions in cell metabolism. Potyviruses use distinct eIF4E isoforms as susceptibility factors in different plants. The role of different members of the plant eIF4E family in the interaction with a given potyvirus could differ drastically. An interplay exists between different members of the eIF4E family in the context of plant-potyvirus interactions, allowing different eIF4E isoforms to modulate each other's availability as susceptibility factors for the virus. In this review, possible molecular mechanisms underlying this interaction are discussed, and approaches to identify the eIF4E isoform that plays a major role in the plant-potyvirus interaction are suggested. The final section of the review discusses how knowledge about the interaction between different eIF4E isoforms can be used to develop plants with durable resistance to potyviruses.

Development of potato (Solanum tuberosum L.) plants with StLEAFY knockout.

MAIN CONCLUSION: StLFY-knockout potato plants were developed using CRISPR/Cas9 system. Inflorescences of edited plants transited to flowering, but inflorescence structures lacked flowers and were indeterminate, producing multiple shoot meristems. The tetraploid potato (Solanum tuberosum L.) is an important agricultural crop worldwide. In this study, we used CRISPR/Cas9 to inactivate the potato homolog (StLFY) of the LEAFY gene-a key regulator of the transition to flowering and floral meristem identity-in a tetraploid potato cultivar. We achieved high rates of all-allelic knockouts. Frameshift indels led to phenotypic alterations, including indeterminate inflorescence development and the replacement of flowers with the leafy-like structures.

VPg of Potato Virus Y and Potato Cap-Binding eIF4E Factors: Selective Interaction and Its Supposed Mechanism.

Potato virus Y (PVY) is one of the most common and harmful plant viruses. Translation of viral RNA starts with the interaction between the plant cap-binding translation initiation factors eIF4E and viral genome-linked protein (VPg) covalently attached to the viral RNA. Disruption of this interaction is one of the natural mechanisms of plant resistance to PVY. The multigene eIF4E family in the potato (Solanum tuberosum L.) genome contains genes for the translation initiation factors eIF4E1, eIF4E2, and eIF(iso)4E. However, which of these factors can be recruited by the PVY, as well as the mechanism of this interaction, remain obscure. Here, we showed that the most common VPg variant from the PVY strain NTN interacts with eIF4E1 and eIF4E2, but not with eIF(iso)4E. Based on the VPg, eIF4E1, and eIF4E2 models and data on the natural polymorphism of VPg amino acid sequence, we suggested that the key role in the recognition of potato cap-binding factors belongs to the R104 residue of VPg. To verify this hypothesis, we created VPg mutants with substitutions at position 104 and examined their ability to interact with potato eIF4E factors. The obtained data were used to build the theoretical model of the VPg-eIF4E2 complex that differs significantly from the earlier models of VPg complexes with eIF4E proteins, but is in a good agreement with the current biochemical data.

An ERF121 transcription factor from Brassica oleracea is a target for the conserved TAL-effectors from different Xanthomonas campestris pv. campestris strains.

Transcription activator-like effectors (TALEs), which induce the expression of specific plant genes to promote infection, are the main pathogenic determinants of various Xanthomonas bacteria. However, investigation of TALEs from Xanthomonas campestris pv. campestris, which causes black rot disease of crucifers, received little attention. In this study, we used PCR-based amplification followed by SMRT amplicon sequencing to identify TALE genes in several X. campestris pv. campestris strains. Computational prediction in conjunction with quantitative reverse transcription PCR analysis was used to find their targets in the Brassica oleracea genome. Transcription factor ERF121, from the AP2/ERF family, was identified as target gene for the conserved TALEs from multiple X. campestris pv. campestris strains. Several members of this family from diverse plants were previously identified as targets of TALEs from different Xanthomonas species. We propose that TALE-dependent activation of AP2/ERF transcription factors promotes susceptibility to Xanthomonas through the misregulation of plant defence pathways.

CRISPR/Cas9 genome editing through in planta transformation.

In this review, the application of CRISPR/Cas9 plant genome editing using alternative transformation methods is discussed. Genome editing by the CRISPR/Cas9 system is usually implemented via the generation of transgenic plants carrying Cas9 and sgRNA genes in the genome. Transgenic plants are usually developed by in vitro regeneration from single transformed cells, which requires using different in vitro culture-based methods. Despite their common application, these methods have some disadvantages and limitations. Thus, some methods of plant transformation that do not depend on in vitro regeneration have been developed. These methods are known as "in planta" transformation. The main focus of this review is the so-called floral dip in planta transformation method, although other approaches are also described. The main features of in planta transformation in the context of CRISPR/Cas9 genome editing are discussed. Furthermore, multiple ways to increase the effectiveness of this approach and to broaden its use in different plant species are considered.

RNA melting and RNA chaperone activities of plant cold shock domain proteins are not correlated.

Cold shock domain proteins (CSDPs) participate in plant development and resistance, but the underlying molecular mechanisms are poorly understood. In this study, we demonstrated that the CSDPs, including EsCSDP1, EsCSDP2, and EsCSDP3, from the extremophyte Eutrema salsugineum possess all basic properties of RNA chaperones. EsCSDP1-3 melt secondary structures in RNAs with various nucleotide sequences and exhibit RNA chaperone activity in vitro. EsCSDP1 and EsCSDP3 were shown to have higher RNA melting activity, whereasile EsCSDP2 had higher RNA chaperone activity. We demonstrated that higher RNA melting activity correlates with the longer C-terminal fragment in many zinc finger motifs, whereas increased RNA chaperone activity was most likely due to the higher glycine content and RGG repeat number in the C-terminal fragment.

High DNA melting activity of extremophyte Eutrema salsugineum cold shock domain proteins EsCSDP1 and EsCSDP3.

Plant cold shock domain proteins (CSDP) participate in maintenance of plant stress tolerance and in regulating their development. In the present paper we show that two out of three extremophyte plant Eutrema salsugineum proteins EsCSDP1-3, namely EsCSDP1 and EsCSDP3, possess high DNA-melting activity. DNA-melting activity of proteins was evaluated using molecular beacon assay in two ways: by measuring Tm parameter (the temperature at which half of the DNA beacon molecules is fully melted) and the beacon fluorescence at 4 °C. As the ratio protein/beacon was increased, a decrease in Tm was observed. Besides DNA-melting activity of full proteins, activity was measured for three isolated cold shock domains EsCSD1-3, C-terminal domain of EsCSDP1 (EsZnF1), as well as a mixture of EsCSD1 and EsZnF1. The Tm reduction efficiency of proteins formed the following sequence: EsCSDP3≈EsCSDP1>(EsCSD1+EsZnF1)>EsZnF1>EsCSDP2. Only full proteins EsCSDP3 and EsCSDP1 demonstrated DNA-melting activity at 4 °C. The presented experimental data indicate that i: interaction of EsCSDP1-3 with beacon single-stranded region is obligatory for efficient melting; ii: cold shock domain and C-terminal domain with zinc finger motifs should be present in one protein molecule to have high melting activity.