Global Analyses of Small Interfering RNAs Derived from Bamboo mosaic virus and Its Associated Satellite RNAs in Different Plants

Background: Satellite RNAs (satRNAs), virus parasites, are exclusively associated with plant virus infection and have attracted much interest over the last 3 decades. Upon virus infection, virus-specific small interfering RNAs (vsiRNAs) are produced by dicer-like (DCL) endoribonucleases for anti-viral defense. The composition of vsiRNAs has been studied extensively; however, studies of satRNA-derived siRNAs (satsiRNAs) or siRNA profiles after satRNA co-infection are limited. Here, we report on the small RNA profiles associated with infection with Bamboo mosaic virus (BaMV) and its two satellite RNAs (satBaMVs) in Nicotiana benthamiana and Arabidopsis thaliana. Methodology/Principal Findings: Leaves of N. benthamiana or A. thaliana inoculated with water, BaMV alone or coinoculated with interfering or noninterfering satBaMV were collected for RNA extraction, then large-scale Solexa sequencing. Up to about 20% of total siRNAs as BaMV-specific siRNAs were accumulated in highly susceptible N. benthamiana leaves inoculated with BaMV alone or co-inoculated with noninterfering satBaMV; however, only about 0.1% of vsiRNAs were produced in plants co-infected with interfering satBaMV. The abundant region of siRNA distribution along BaMV and satBaMV genomes differed by host but not by co-infection with satBaMV. Most of the BaMV and satBaMV siRNAs were 21 or 22 nt, of both (+) and (-) polarities; however, a higher proportion of 22-nt BaMV and satBaMV siRNAs were generated in N. benthamiana than in A. thaliana. Furthermore, the proportion of non-viral 24-nt siRNAs was greatly increased in N. benthamiana after virus infection. Conclusions/Significance: The overall composition of vsiRNAs and satsiRNAs in the infected plants reflect the combined action of virus, satRNA and different DCLs in host plants. Our findings suggest that the structure and/or sequence demands of various DCLs in different hosts may result in differential susceptibility to the same virus. DCL2 producing 24-nt siRNAs under biotic stresses may play a vital role in the antiviral mechanism in N. benthamiana

A <i>De Novo</i> Floral Transcriptome Reveals Clues into <i>Phalaenopsis</i> Orchid Flower Development

<div><p><i>Phalaenopsis</i> has a zygomorphic floral structure, including three outer tepals, two lateral inner tepals and a highly modified inner median tepal called labellum or lip; however, the regulation of its organ development remains unelucidated. We generated RNA-seq reads with the Illumina platform for floral organs of the <i>Phalaenopsis</i> wild-type and peloric mutant with a lip-like petal. A total of 43,552 contigs were obtained after <i>de novo</i> assembly. We used differentially expressed gene profiling to compare the transcriptional changes in floral organs for both the wild-type and peloric mutant. Pair-wise comparison of sepals, petals and labellum between peloric mutant and its wild-type revealed 1,838, 758 and 1,147 contigs, respectively, with significant differential expression. <i>PhAGL6a</i> (CUFF.17763), <i>PhAGL6b</i> (CUFF.17763.1), <i>PhMADS1</i> (CUFF.36625.1), <i>PhMADS4</i> (CUFF.25909) and <i>PhMADS5</i> (CUFF.39479.1) were significantly upregulated in the lip-like petal of the peloric mutant. We used real-time PCR analysis of lip-like petals, lip-like sepals and the big lip of peloric mutants to confirm the five genes’ expression patterns. <i>PhAGL6a</i>, <i>PhAGL6b</i> and <i>PhMADS4</i> were strongly expressed in the labellum and significantly upregulated in lip-like petals and lip-like sepals of peloric-mutant flowers. In addition, <i>PhAGL6b</i> was significantly downregulated in the labellum of the big lip mutant, with no change in expression of <i>PhAGL6a</i>. We provide a comprehensive transcript profile and functional analysis of <i>Phalaenopsis</i> floral organs. <i>PhAGL6a PhAGL6b</i>, and <i>PhMADS4</i> might play crucial roles in the development of the labellum in <i>Phalaenopsis</i>. Our study provides new insights into how the orchid labellum differs and why the petal or sepal converts to a labellum in <i>Phalaenopsis</i> floral mutants.</p></div

The development of functional mapping by three sex-related loci on the third whorl of different sex types of <i>Carica papaya</i> L.

<div><p><i>Carica papaya</i> L. is an important economic crop worldwide and is used as a model plant for sex-determination research. To study the different flower sex types, we screened sex-related genes using alternative splicing sequences (AS-seqs) from a transcriptome database of the three flower sex types, i.e., males, females, and hermaphrodites, established at 28 days before flowering using 15 bacterial artificial chromosomes (BACs) of <i>C</i>. <i>papaya</i> L. After screening, the cDNA regions of the three sex-related loci, including short vegetative phase-like (<i>CpSVPL</i>), the chromatin assembly factor 1 subunit A-like (<i>CpCAF1AL</i>), and the somatic embryogenesis receptor kinase (<i>CpSERK</i>), which contained eight sex-related single-nucleotide polymorphisms (SNPs) from the different sex types of <i>C</i>. <i>papaya</i> L., were genotyped using high-resolution melting (HRM). The three loci were examined regarding the profiles of the third whorl, as described below. <i>CpSVPL</i>, which had one SNP associated with the three sex genotypes, was highly expressed in the male and female sterile flowers (abnormal hermaphrodite flowers) that lacked the fourth whorl structure. <i>CpCAF1AL</i>, which had three SNPs associated with the male genotype, was highly expressed in male and normal hermaphrodite flowers, and had no AS-seqs, whereas it exhibited low expression and an AS-seqs in intron 11 in abnormal hermaphrodite flowers. Conversely, carpellate flowers (abnormal hermaphrodite flowers) showed low expression of <i>CpSVPL</i> and AS-seqs in introns 5, 6, and 7 of <i>CpSERK</i>, which contained four SNPs associated with the female genotype. Specifically, the <i>CpSERK</i> and <i>CpCAF1AL</i> loci exhibited no AS-seq expression in the third whorl of the male and normal hermaphrodite flowers, respectively, and variance in the AS-seq expression of all other types of flowers. Functional mapping of the third whorl of normal hermaphrodites indicated no AS-seq expression in <i>CpSERK</i>, low <i>CpSVPL</i> expression, and, for <i>CpCAF1A</i>L, high expression and no AS-seq expression on XY^h-type chromosomes.</p></div

Possible evolutionary relationships between <i>PhAGL6a</i>, <i>PhAGL6b</i> and <i>PhMADS4</i> in the regulation of lip formation in <i>Phalaenopsis</i> orchid.

<p>Possible evolutionary relationships between <i>PhAGL6a</i>, <i>PhAGL6b</i> and <i>PhMADS4</i> in the regulation of lip formation in <i>Phalaenopsis</i> orchid.</p

Annotation of the <i>Phalaenopsis</i> transcriptome by gene ontology (GO), KEGG and Pfam classification.

<p>(A) GO classification summarized by three main categories: biological process, cellular component and molecular function. (B) Functional annotation of transcripts based on KEGG classification. (C) Functional characterization of transcripts for enzyme classes. (D) Pfam domains identified in translated <i>Phalaenopsis</i> transcripts.</p

Summary of <i>Phalaenopsis</i> floral-organ transcriptome assembly.

<p>NS, wild-type sepal; PS, peloric sepal; NP, wild-type petal; PP, peloric petal; NL, wild-type labellum; PL, peloric labellum (PL)</p><p>* All reads mixed.</p><p>Summary of <i>Phalaenopsis</i> floral-organ transcriptome assembly.</p

Flowers of wild-type and peloric mutant of <i>Phalaenopsis</i> Brother Spring Dancer ‘KHM190’.

<p>(A) Wild-type and (B) peloric mutant flower. Bar = 1 cm. (C) Scanning electron microscopy of petal of floral buds at early developmental stages of (a), (b) wild-type and (c), (d) peloric-mutant flower. Bar = 500 μm.</p