Evolution of Plant Male Germline-Specific Transcription Factor DUO POLLEN 1

Flowering plants account for the 30 crops that provide 95 % of the food for humans. The reproduction of this group depends on the production of two twin sperms. The establishment of the male germline lineage requires the transcription factor DUO POLLEN 1 (DUO1). DUO1 is required for both the cell cycle progression and sperm cell differentiation. This thesis focused on the origin of DUO1 and its target regulation. Much work was dedicated in searching the evolutionary origin of DUO1 in the R2R3 MYB clade. Based on the analysis of sequences homologous to DUO1 and its sister clade GAMYB, the earliest DUO1 homolog was identified in the green algae. The DUO1 clade did not proliferate after multiple polyploidy events, possibly restricted by its male germline-specific role supported by transcriptome data. The ancestral DUO1 experienced a major MYB domain sequence change in the bryophytes and a second change in the C-terminus in the angiosperms. The MYB domain changes caused a change in the target DNA sequence, which has then been conserved among Embryophyta DUO1 homologs. Another change also happened in the region where a miR159 binding site is present in most angiosperm DUO1 homologs. Sequence and functional analysis showed that this change evolved long before the emergence of miR159. The changes in the C-terminus of DUO1 led to a higher target promoter activation capability in the angiosperm homologs, which was confirmed by functional tests of the angiosperm and bryophyte DUO1. This C-terminal region contains the transactivation domain (TAD) of DUO1 and certain functionally important motifs were highlighted in the study. While these motifs indicated that DUO1 was a member of a TAD family, it was also demonstrated that unknown sequences carry critical features for activation. Together these results mapped the evolution history of DUO1 in the Streptophyta lineage

Assorted Processing of Synthetic Trans-Acting siRNAs and Its Activity in Antiviral Resistance

<div><p>The use of syn-tasiRNAs has been proposed as an RNA interference technique alternative to those previously described: hairpin based, virus induced gene silencing or artificial miRNAs. In this study we engineered the TAS1c locus to impair <i>Plum pox virus</i> (PPV) infection by replacing the five native siRNAs with two 210-bp fragments from the CP and the 3´NCR regions of the PPV genome. Deep sequencing analysis of the small RNA species produced by both constructs <i>in planta</i> has shown that phased processing of the syn-tasiRNAs is construct-specific. While in syn-tasiR-CP construct the processing was as predicted 21-nt phased in register with miR173-guided cleavage, the processing of syn-tasiR-3NCR is far from what was expected. A 22-nt species from the miR173-guided cleavage was a guide of two series of phased small RNAs, one of them in an exact 21-nt register, and the other one in a mixed of 21-/22-nt frame. In addition, both constructs produced abundant PPV-derived small RNAs in the absence of miR173 as a consequence of a strong sense post-transcriptional gene silencing induction. The antiviral effect of both constructs was also evaluated in the presence or absence of miR173 and showed that the impairment of PPV infection was not significantly higher when miR173 was present. The results show that syn-tasiRNAs processing depends on construct-specific factors that should be further studied before the so-called MIGS (miRNA-induced gene silencing) technology can be used reliably.</p></div

In-phase processing of syn-tasiRNA precursors directed by miR173.

<p>Phased syn-tasiRNAs (21 nt) (D1-D7) in register with the miR173-guided cleavage site (indicated with an arrow) from syn-tasiR-CP <b>(A)</b> and syn-tasiR-3NCR <b>(B)</b> constructs. <b>(C)</b> Two series of phased syn-tasiRNAs (21 and 22 nt) in register with the miR173-guided cleavage site (indicated with an arrow). One series is marked as a-aa and the second one as a-ab. Numbers above and below the syn-tasiRNAs indicate reads from samples syn-tasiR+173/syn-tasiR-173. The mi173 target site (in purple) and the PPV sequences are shown in bold.</p

Syn-tasiRNA constructs and miR173 accumulation in <i>N</i>. <i>benthamiana</i> transient assays.

<p><b>A)</b> Diagram of TAS1-derived syn-tasi constructs containing PPV sequences. The tasiRNA-spawning region is indicated by brackets and the length of the PPV regions included in the constructs is shown. The miR173 target site is indicated by a line. <b>B)</b> Agroinfiltration transient assay in <i>N</i>. <i>benthamiana</i>. Syn-tasiRNA constructs were expressed individually or in combination with miR173 precursor. <b>C)</b> Blot assay to assess the accumulation of miR173 in the plant tissue three days after agroinfiltration. Two biological replicates are shown. EtBr-stained 5S rRNA/tRNA is shown.</p

Antiviral effect of PPV-specific syn-tasiRNA constructs.

<p><b>(A</b> and <b>B</b>) The indicated combinations of MIR173 (miR173), MIR159 (miR159), empty pMDC32 (vector) syn-tasiR-3NCR and syn-tasiR-CP were transiently expressed by agroinfiltration in <i>N</i>. <i>benthamiana</i> plants. Three days post-agroinfiltration plants were inoculated with PPV-GFP and 5 days post-inoculation (dpi) infection foci were observed under a fluorescence microscope. Number of foci/leaves is indicated. <b>(C)</b> Agroinfiltrated leaves harvested at 5 dpi were subjected to immunoblot analysis with anti-CP serum. The membrane stained with Ponceau red is included as loading control.</p

Effect of miR173 expression in size distribution profiles of syn-tasiRNAs produced in <i>N</i>. <i>benthamiana</i> transient assays.

<p>Size distribution of small RNAs (18–26 nt) identified by high-throughput sequencing is shown. <b>A)</b> Small RNAs mapping to <i>N</i>. <i>benthamiana</i> genome. <b>B)</b> Small RNAs mapping to the common region of pMDC32-derived plasmids. <b>C)</b> Small RNAs mapping to PPV-derived regions from syn-tasiR-3NCR (3NCR) and syn-tasiR-CP (CP) constructs.</p

Effect of miR173 expression for syn-tasiRNA accumulation in <i>N</i>. <i>benthamiana</i> transient assays.

<p>Mapping of small RNA sequences obtained by high-throughput sequencing are shown. Analyses of total small RNA (18–26 nt) reads in the presence (+miR173) or absence of miR173 (-miR173) is shown in percentages in pie charts. Those reads mapping across the syn-tasiRNA constructs (construct specific) are shown in detail. CP: reads mapping to CP region included in the syn-tasiR-CP. 3NCR: reads mapping to 3’NCR region included in the syn-tasiR-3NCR. Ath-MIR173: reads mapping to the MIR173 construct.</p

Table_1_Transcriptome analysis of genes involved in the pathogenesis mechanism of potato virus Y in potato cultivar YouJin.XLSX

IntroductionPotatoes (Solanum tuberosum L.) can be infected by various viruses, but out of all of viruses, the potato virus Y (PVY) is the most detrimental. Research shows that the potato cultivar YouJin is especially vulnerable to PVY and displays severe symptoms, including leaf vein chlorosis, curled leaf margins, large necrotic spots on the leaf blades, and the growth of small new leaves.MethodsPVY infection in potato cultivar YouJin was confirmed through symptom observation, RT-PCR, and Western blot analysis. Transcriptome sequencing was used to analyze the genes associated with PVY pathogenesis in this cultivar.ResultTranscriptome analysis of differential genes was conducted in this study to examine the pathogenesis of PVY on YouJin. The results showed that 1,949 genes were differentially regulated, including 853 upregulated genes and 1,096 downregulated genes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that carbohydrate synthesis and metabolism pathways were suppressed, and electron transferase and hydrolase activities were reduced. Moreover, there were increased expression levels of protein kinase genes. By focusing on plant–pathogen interaction pathways, six core genes all upregulating the WARK family of transcription factors were obtained. Additionally, a constructed PPI network revealed the identification of key modular differential genes, such as downregulated photosynthesis-related protein genes and upregulated AP2/ERF-ERF transcription factors. Functional network enrichment analysis revealed that PVY infection limited RNA metabolism, glutathionylation, and peroxiredoxin activity while triggering the expression of associated defense genes in YouJin. After analyzing the above, 26 DEGs were screened and 12 DEGs were confirmed via RT-qPCR.ConclusionThese results establish a hypothetical framework for clarifying the pathogenesis of PVY in the YouJin variety of potatoes, which will help design the disease resistance of YouJin.</p

Table_7_Transcriptome analysis of genes involved in the pathogenesis mechanism of potato virus Y in potato cultivar YouJin.XLSX

IntroductionPotatoes (Solanum tuberosum L.) can be infected by various viruses, but out of all of viruses, the potato virus Y (PVY) is the most detrimental. Research shows that the potato cultivar YouJin is especially vulnerable to PVY and displays severe symptoms, including leaf vein chlorosis, curled leaf margins, large necrotic spots on the leaf blades, and the growth of small new leaves.MethodsPVY infection in potato cultivar YouJin was confirmed through symptom observation, RT-PCR, and Western blot analysis. Transcriptome sequencing was used to analyze the genes associated with PVY pathogenesis in this cultivar.ResultTranscriptome analysis of differential genes was conducted in this study to examine the pathogenesis of PVY on YouJin. The results showed that 1,949 genes were differentially regulated, including 853 upregulated genes and 1,096 downregulated genes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that carbohydrate synthesis and metabolism pathways were suppressed, and electron transferase and hydrolase activities were reduced. Moreover, there were increased expression levels of protein kinase genes. By focusing on plant–pathogen interaction pathways, six core genes all upregulating the WARK family of transcription factors were obtained. Additionally, a constructed PPI network revealed the identification of key modular differential genes, such as downregulated photosynthesis-related protein genes and upregulated AP2/ERF-ERF transcription factors. Functional network enrichment analysis revealed that PVY infection limited RNA metabolism, glutathionylation, and peroxiredoxin activity while triggering the expression of associated defense genes in YouJin. After analyzing the above, 26 DEGs were screened and 12 DEGs were confirmed via RT-qPCR.ConclusionThese results establish a hypothetical framework for clarifying the pathogenesis of PVY in the YouJin variety of potatoes, which will help design the disease resistance of YouJin.</p

MYB81, a microspore-specific GAMYB transcription factor, promotes pollen mitosis I and cell lineage formation in Arabidopsis.

Sexual reproduction in flowering plants relies on the production of haploid gametophytes that consist of germline and supporting cells. During male gametophyte development, the asymmetric mitotic division of an undetermined unicellular microspore segregates these two cell lineages. To explore genetic regulation underlying this process, we screened for pollen cell patterning mutants and isolated the heterozygous myb81-1 mutant that sheds ~50 % abnormal pollen. Typically, myb81-1 microspores fail to undergo pollen mitosis I and arrest at polarized stage with a single central vacuole. Although most myb81-1 microspores degenerate without division, a small fraction divide at later stages and fail to acquire correct cell fates. The myb81-1 allele is transmitted normally through the female, but rarely through pollen. We show that myb81-1 phenotypes result from impaired function of the GAMYB transcription factor MYB81. The MYB81 promoter shows microspore-specific activity and a MYB81-RFP fusion protein is only expressed in a narrow window prior to pollen mitosis I. Ectopic expression of MYB81 driven by various promoters can severely impair vegetative or reproductive development, reflecting the strict microspore-specific control of MYB81. Our data demonstrate that MYB81 has a key role in the developmental progression of microspores, enabling formation of the two male cell lineages that are essential for sexual reproduction in Arabidopsis