Virus versus Host Plant MicroRNAs: Who Determines the Outcome of the Interaction?

<div><p>Considering the importance of microRNAs (miRNAs) in the regulation of essential processes in plant pathogen interactions, it is not surprising that, while plant miRNA sequences counteract viral attack via antiviral RNA silencing, viruses in turn have developed antihost defense mechanisms blocking these RNA silencing pathways and establish a counter-defense. In the current study, computational and stem-loop Reverse Transcription – Polymerase Chain Reaction (RT-PCR) approaches were employed to a) predict and validate virus encoded mature miRNAs (miRs) in 39 DNA-A sequences of the bipartite genomes of <i>African cassava mosaic virus</i> (ACMV) and <i>East African cassava mosaic virus</i>-Uganda (EACMV-UG) isolates, b) determine whether virus encoded miRs/miRs* generated from the 5′/3′ harpin arms have the capacity to bind to genomic sequences of the host plants <i>Jatropha</i> or cassava and c) investigate whether plant encoded miR/miR* sequences have the potential to bind to the viral genomes. Different viral pre-miRNA hairpin sequences and viral miR/miR* length variants occurring as isomiRs were predicted in both viruses. These miRNAs were located in three Open Reading Frames (ORFs) and in the Intergenic Region (IR). Moreover, various target genes for miRNAs from both viruses were predicted and annotated in the host plant genomes indicating that they are involved in biotic response, metabolic pathways and transcription factors. Plant miRs/miRs* from conserved and highly expressed families were identified, which were shown to have potential targets in the genome of both begomoviruses, representing potential plant miRNAs mediating antiviral defense. This is the first assessment of predicted viral miRs/miRs* of ACMV and EACMV-UG and host plant miRNAs, providing a reference point for miRNA identification in pathogens and their hosts. These findings will improve the understanding of host- pathogen interaction pathways and the function of viral miRNAs in <i>Euphorbiaceous</i> crop plants.</p></div

Fourteen viral pre-miRNAhairpins from 11 ACMV and 28 EACMV-UG isolates from <i>Jatropha</i>and cassava were classified as real or pseudo.

<p>The number of virus sequences, their locationin the virus genome and their hairpin sequences are shown.</p><p>*position based on [Genbank:JN053428 and JN053454].</p

Sequences of DNA A of 11 ACMV and 28 EACMV-UG deposited in the Genbank were compared [4].

<p>The sequences were isolated from <i>Jatropha</i> and cassava (in bold).</p

End point PCR amplification of ACMV and EACMV-UG virus miRNAs.

<p>PCR products of 60-infected with ACMV and EACMV: S2C6, S4C6, –RT control. Actin (76 bp) was used as internal control.</p

Nucleotide content of 7 real ACMV and 2 real EACMV-UG miRNA hairpins.

<p>Nucleotide content of 7 real ACMV and 2 real EACMV-UG miRNA hairpins.</p

End point PCR amplification of plant miRNAs on cassava and <i>Jatropha</i>.

<p>PCR products of 60-infected cassava plant samples, respectively: S4C4, S2C6, S4C6, B2C15, –RT control. Lanes 6–7 are one infected and one non-infected <i>Jatropha</i> plant samples, respectively: K5J5, S4J12. <i>Actin</i> (76 bp) was used as internal control.</p

Secondary structures of 9 predicted real viral pre-miRNA hairpins.

<p>The secondary structures of the real pre-miRNAs were folded using the RNAshapes.</p

The novel virus miR/miR* sequences predicted from ACMV and EACMV-UG real pre-miRNA hairpins.

<p>The miR/miRs* were predicted using <i>Jatropha</i> and cassava sequence hits from BlastN. The length, location on 5′ or 3′ arms of hairpins and sequence of the nucleotides 2–8 at the 5′ end (highlighted in bold) using RNAShape are shown.</p

Outline of the strategy to identify plant miRNAs from miRBase that potentially target regions of DNA-A in ACMV and EACMV-UG.

<p>Computational approaches were identified plant miRs/miRs* with potential to target viral genomic regions.</p

Outline of the strategy to identify miRNAs in viral DNA-A of ACMV and EACMV-UG and their potential targets in the host plants <i>Jatropha</i> and cassava.

<p>Computational approaches were used to scan and filter the DNA-A genomes of 11 ACMV and 28 EACMV-UG isolates to identify novel virus miRNAs and their targets in <i>Jatropha</i> and cassava.</p