Structure alignment of Rrp4 trimeric cap between our <i>S. solfataricus</i> exosome (red) and that Lorentzen <i>et al</i> previously reported [30] (cyan).

<p>(<i>a</i>) Overall alignment of the 750− aa trimeric cap (r.m.s.d. = 1.5 Å). (<i>b</i>) Alignment of the most flexible Rrp4 subunit (Chain F) by N-ter domain, aa 1–50 (r.m.s.d. = 0.4 Å). (<i>c</i>) Alignment of Chain F subunit by S1 domain, aa 56–126 (r.m.s.d. = 0.3 Å). (<i>d</i>) Alignment of Chain F subunit by KH domain, aa 135–250 (r.m.s.d. = 0.7 Å).</p

Detecting thermal motions in Rrp4 RNA-binding ring using thermal ellipsoid and B-factor analyses.

<p>(<i>a</i>) Overall analysis of the trimeric cap. (<i>b</i>) According to the thermal ellipsoid analysis, the most thermal flexible Rrp4 subunit is Chain I, but not Chain F, which displays the largest rigidbody motion. TLS sensors were obtained from TLS refinement in Refmac5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008739#pone.0008739-Collaborative1" target="_blank">[48]</a> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008739#s4" target="_blank">Methods</a> section for details) and plotted using Raster3D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008739#pone.0008739-Merritt1" target="_blank">[51]</a>.(<i>c</i>) B-factor comparison of the Rrp4 between our <i>S. solfataricus</i> exosome (left) and that Lorentzen <i>et al</i> previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008739#pone.0008739-Lorentzen2" target="_blank">[30]</a> (right). B-factor coloring: blue, 30 and below; red, 100 and above.</p

Rrp4 trimeric cap between our <i>S. solfataricus</i> exosome (blue) deviates from perfect three-fold symmetry as compared with the structure by Lorentzen <i>et al</i>[30] (orange).

<p>Subunit F and I considerably deviates (2 to 3 Å at the periphery) from Lorentzen <i>et al</i> previously report when aligning subunit C. Inlets: 2.9 Å experimental electron density map of Rrp4 subunit F contoured at 1.0 σ.</p

Crystallographic statistics.

<p>Crystallographic statistics.</p

Purification and activity assay of the purified <i>S. solfataricus</i> full exosome.

<p>(<i>a</i>) SDS-PAGE of the purified <i>S. solfataricus</i> full exosome (left) and the Rrp4-exosome isoform (right). (b) RNase activity assay for the intact <i>S. solfataricus</i> exosome. The exosome is stalled by the HDV ribozyme sequence. (<i>c</i>) Side and top-down view of the 2.9 Å <i>S. solfataricus</i> exosome structure. Gold, trimeric Rrp4 RNA-binding ring; blue/cyan, Rrp41/Rrp42 catalytic ring.</p

Localization and distribution of CaLas in citrus roots.

<p>A-H: CaLas in roots collected from HLB-symptomatic ‘Valencia’ orange tree 1. I-K: CaLas in roots collected from HLB-symptomatic ‘Valencia’ orange 2. L: Healthy sweet orange. M, N: Roots of ‘Valencia’ orange trees 1 and 2.</p

Irregular distribution of CaLas in phloem sieve cells.

<p>A-D: Localized infection of phloem cells by CaLas and E-K: generalized infection of phloem cells by CaLas. L: healthy sweet orange control, sampled at the midpoint between the abscission zones.</p

Localization and Distribution of '<i>Candidatus</i> Liberibacter asiaticus’ in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody

<div><p>‘<i>Candidatus</i> Liberibacter asiaticus’ (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant.</p></div

Localization and distribution of CaLas in sweet orange seed.

<p>A-G: CaLas infected and symptomatic ‘Valencia’ seeds collected from symptomatic fruit in Florida. H-K: Sweet orange seed from Florida (asymptomatic commercial fruit from supermarket). L: Healthy sweet orange seed.</p

Visual symptoms in periwinkle leaves used for the DTBIA in Fig 8 with the Cq values for relative CaLas concentrations estimated by qPCR.

<p>Visual symptoms in periwinkle leaves used for the DTBIA in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123939#pone.0123939.g008" target="_blank">Fig 8</a> with the Cq values for relative CaLas concentrations estimated by qPCR.</p