Integrated transcriptome and microRNA sequencing analyses reveal gene responses in poplar leaves infected by the novel pathogen bean common mosaic virus (BCMV).

Recently, a novel poplar mosaic disease caused by bean common mosaic virus (BCMV) was investigated in Populus alba var. pyramidalis in China. Symptom characteristics, physiological performance of the host, histopathology, genome sequences and vectors, and gene regulation at the transcriptional and posttranscriptional levels were analyzed and RT-qPCR (quantitative reverse transcription PCR) validation of expression was performed in our experiments. In this work, the mechanisms by which the BCMV pathogen impacts physiological performance and the molecular mechanisms of the poplar response to viral infection were reported. The results showed that BCMV infection decreased the chlorophyll content, inhibited the net photosynthesis rate (Pn) and stomatal conductance (Gs), and significantly changed chlorophyll fluorescence parameters in diseased leaves. Transcriptome analysis revealed that the expression of the majority of DEGs (differentially expressed genes) involved in the flavonoid biosynthesis pathway was promoted, but the expression of all or almost all DEGs associated with photosynthesis-antenna proteins and the photosynthesis pathway was inhibited in poplar leaves, suggesting that BCMV infection increased the accumulation of flavonoids but decreased photosynthesis in hosts. Gene set enrichment analysis (GSEA) illustrated that viral infection promoted the expression of genes involved in the defense response or plant-pathogen interaction. MicroRNA-seq analysis illustrated that 10 miRNA families were upregulated while 6 families were downregulated in diseased poplar leaves; moreover, miR156, the largest family with the most miRNA members and target genes, was only differentially upregulated in long-period disease (LD) poplar leaves. Integrated transcriptome and miRNA-seq analyses revealed 29 and 145 candidate miRNA-target gene pairs; however, only 17 and 76 pairs, accounting for 2.2% and 3.2% of all DEGs, were authentically negatively regulated in short-period disease (SD) and LD leaves, respectively. Interestingly, 4 miR156/SPL (squamosa promoter-binding-like protein) miRNA-target gene pairs were identified in LD leaves: the miR156 molecules were upregulated, but SPL genes were downregulated. In conclusion, BCMV infection significantly changed transcriptional and posttranscriptional gene expression in poplar leaves, inhibited photosynthesis, increased the accumulation of flavonoids, induced systematic mosaic symptoms, and decreased physiological performance in diseased poplar leaves. This study elucidated the fine-tuned regulation of poplar gene expression by BCMV; moreover, the results also suggested that miR156/SPL modules played important roles in the virus response and development of viral systematic symptoms in plant virus disease.

The C2 H2 -type zinc finger transcription factor OSIC1 positively regulates stomatal closure under osmotic stress in poplar.

Salt and drought impair plant osmotic homeostasis and greatly limit plant growth and development. Plants decrease stomatal aperture to reduce water loss and maintain osmotic homeostasis, leading to improved stress tolerance. Herein, we identified the C2 H2 transcription factor gene OSMOTIC STRESS INDUCED C2 H2 1 (OSIC1) from Populus alba var. pyramidalis to be induced by salt, drought, polyethylene glycol 6000 (PEG6000) and abscisic acid (ABA). Overexpression of OSIC1 conferred transgenic poplar more tolerance to high salinity, drought and PEG6000 treatment by reducing stomatal aperture, while its mutant generated by the CRISPR/Cas9 system showed the opposite phenotype. Furthermore, OSIC1 directly up-regulates PalCuAOζ in vitro and in vivo, encoding a copper-containing polyamine oxidase, to enhance H2 O2 accumulation in guard cells and thus modulates stomatal closure when stresses occur. Additionally, ABA-, drought- and salt-induced PalMPK3 phosphorylates OSIC1 to increase its transcriptional activity to PalCuAOζ. This regulation of OSIC1 at the transcriptional and protein levels guarantees rapid stomatal closure when poplar responds to osmotic stress. Our results revealed a novel transcriptional regulatory mechanism of H2 O2 production in guard cells mediated by the OSIC1-PalCuAOζ module. These findings deepen our understanding of how perennial woody plants, like poplar, respond to osmotic stress caused by salt and drought and provide potential targets for breeding.

Bacillus velezensis BY6 Promotes Growth of Poplar and Improves Resistance Contributing to the Biocontrol of Armillaria solidipes.

To improve the application of endophyte Bacillus velezensis BY6 from the xylem of poplar, the effect of BY6 on the growth of diseased Populus davidiana × Populus. alba var. pyramidalis Louche (Pdpap poplar) seedlings and the biological control effect on the pathogen Armillaria solidipes were tested using a plant split-root experiment. After applying BY6 to the roots of diseased Pdpap poplar seedlings, the results show that plant growth indicators (dry mass, fresh mass, and plant height) were significantly increased (p < 0.05), and genes related to auxin hormone signal transcription were activated. BY6 indicated a surprising control effect after the inoculation of diseased Pdpap poplar seedlings. Compared to the infected control group, the treated disease index of the diseased Pdpap poplar seedlings in the treatment group were reduced by 49.53% on the 20th day. The relative staining areas of diaminobenzidine (DAB) and Trypan blue decreased by 3.37 and 7.31 times, respectively. The physiological indicators (soluble sugar and protein) and oxidase indicators were significantly increased (p < 0.05). The expression levels of defense genes related to salicylic acid (SA) and jasmonic acid (JA) signaling pathways were significantly increased (p < 0.05). Amazingly, the results indicate that BY6 simultaneously activates induced systemic resistance (ISR) and systemic acquired resistance (SAR) in diseased Pdpap poplar seedlings and promotes growth. The results indicate that BY6 is a promising candidate for developing forest tree biofertilizers and biopesticides.

Somatic mutations during rapid clonal domestication of Populus alba var. pyramidalis.

For many clonally propagated species, the accumulation of somatic mutations is the principal driver of declines in yield and quality. However, somatic mutations may also promote genetic diversification. Thus, elucidating somatic mutation rates and patterns is important to understand the genetic basis undergirding the emergence of commercially valuable traits and developmental processes. In this study, we studied the effect of short-time clonal domestication of Populus alba var. pyramidalis, a species that has been propagated by cutting for the last 67 years. We found that: (1) the somatic mutation rate for P. alba var. pyramidalis is 9.24 × 10-9, which is higher than rates observed in related species; (2) there were more mutations near heterozygous regions, and a larger proportion of CpG and CHG sites were associated with somatic mutations, which may be related to the blocking of DNA repair by methylation; and (3) deleterious mutations were not shared by multiple individuals, and all occurred in heterozygous states, demonstrating the strong selective pressures that act against deleterious mutations. Taken together, the results of our study provide a global view of somatic mutation that will aid efforts to understand the genetic basis of commercially valuable traits and to improve clonally breeding species.

Analysis of Alternative Splicing and Alternative Polyadenylation in Populus alba var. pyramidalis by Single-Molecular Long-Read Sequencing.

Poplars are worldwidely cultivated with ecologically and economically important value. Populus alba var. pyramidalis (= P. bolleana) is a main tree of the farmland shelter-belt system in the arid region of Northwest China due to its rapid growth, erect stems, and high biomass production. However, the full-length messenger RNA (mRNA) sequences and complete structure of P. alba var. pyramidalis remain unclear. In this study, using single-molecular real-time (SMRT) and next-generation high-throughput sequencing (NGS) platform, we sequenced transcripts from leaf, root, xylem, and phloem of P. alba var. pyramidalis, to obtain the full-length mRNA transcripts and annotate the complete structure. In total, 86,327 mapped full-length non-chimeric (FLNC) reads were identified, with 705 previously unannotated loci and 3,410 long noncoding RNAs (lncRNAs) and 174 fusion genes found. Alternative spicing (AS) events were detected in 7,536 genes, of which 4,652 genes had multiple AS events. A total of 10,213 alternative polyadenylation (APA) sites were identified, with two or more APA sites observed in 2,212 genes. Our transcriptome data provided the full-length sequences and gene isoforms of transcripts for P. alba var. pyramidalis, which will be helpful in improving our understanding for the genome annotation and gene structures of P. alba var. pyramidalis.

The PalERF109 transcription factor positively regulates salt tolerance via PalHKT1;2 in Populus alba var. pyramidalis.

Salinity restricts the growth of trees to varying extents, but the regulatory mechanisms involved in their varying salt tolerance are largely unknown. In an effort to elucidate these mechanisms, we identified a total of 99 genes in the Ethylene Responsive Factor (ERF) family of transcription factors and examined their expression patterns under salt stress in Populus alba var. pyramidalis. We found that a B4 group gene, PalERF109, was rapidly induced by salt treatment and preferentially expressed in stems and petioles, where it is probably involved in transport of ions and water in xylem. Overexpression of PalERF109 enhanced the salt tolerance of the poplar, and further analysis showed that it directly upregulated a high-affinity K+transporter (HKT) gene, PalHKT1;2. The results clearly indicate that PalERF109 enhances salt tolerance at least partially through direct activation of PalHKT1;2 and extends understanding of the roles of ERF genes in tree stress responses.

Coexpression of PalbHLH1 and PalMYB90 Genes From Populus alba Enhances Pathogen Resistance in Poplar by Increasing the Flavonoid Content.

Secondary metabolites of the flavonoid pathway participate in plant defense, and bHLH and MYB transcription factors regulate the synthesis of these metabolites. Here, we define the regulatory mechanisms in response to pathogens. Two transcription factors from Populus alba var. pyramidalis, PalbHLH1 and PalMYB90, were overexpressed together in poplar, and transcriptome analysis revealed differences in response to pathogen infection. The transgenic plants showed elevated levels of several key flavonoid pathway components: total phenols, proanthocyanidins (PAs), and anthocyanins and intermediates quercetin and kaempferol. Furthermore, PalbHLH1 and PalMYB90 overexpression in poplar enhanced antioxidase activities and H2O2 release and also increased resistance to Botrytis cinerea and Dothiorella gregaria infection. Gene expression profile analysis showed most genes involved in the flavonoid biosynthesis pathway or antioxidant response to be upregulated in MYB90/bHLH1-OE poplar, but significant differential expression occurred in response to pathogen infection. Specifically, expression of PalF3H (flavanone 3-hydroxylase), PalDFR (dihydroflavonol 4-seductase), PalANS (anthocyanin synthase), and PalANR (anthocyanin reductase), which function in initial, middle, and final steps of anthocyanin and PA biosynthesis, respectively, was significantly upregulated in D. gregaria-infected MYB90/bHLH1-OE poplar. Our results highlight that PalbHLH1 and PalMYB90 function as transcriptional activators of flavonoid pathway secondary-metabolite synthesis genes, with differential mechanisms in response to bacterial or fungal infection.