Reference maps from 2-DE analysis of conidial proteins from <i>F</i>. <i>oxysporum</i> f. sp <i>conglutinans</i>.

<p>Proteins were separated by using 24 cm, pH 4–7 IPG strips for IEF and followed by 12.5% SDS-PAGE. The gels were stained by GAP method, which used CBB-G250, ammonium sulfate, and phosphoric acid. A: Gel of conidia proteins from Foc grown in PDB. B: The four proteins (spots 1, 2, 3 and 4), named Foc-SIX1 with high abundance in Foc, were indicated by arrows and numbers and listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152273#pone.0152273.t001" target="_blank">Table 1</a>; Figure B is the partial enlarged details of the rectangular box in Figure A.</p

The expression of <i>SIX1</i> gene in mycelia, conidia and infected cabbage roots at 7 dpi.

<p>Mycelia and conidia were obtained from Foc grown in PDB for 48 h. Gene expression levels were relative to the internal controls <i>EF-1α</i> and <i>β-tubulin</i>. Averages of four biological replicates were shown with the standard error.</p

Phylogenetic analysis of Foc-SIX1 by using neighbor-joining method.

<p>The tree was generated based on alignments of full amino acid sequences of homologs using MEGA 6 with 1000 bootstraps.</p

Identification of the four proteins indicated in Fig 1 from the conidia of Foc.

<p>Identification of the four proteins indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152273#pone.0152273.g001" target="_blank">Fig 1</a> from the conidia of Foc.</p

Construction, verification, and virulence analysis of complementation mutants Foc-ΔSIX1::Foc-SIX1.

<p>A: Foc-ΔSIX1 mutants were complemented by transformation with approximately 3.0 kb of <i>Foc-SIX1</i> spanning from -1838 bp (promoter relative to start codon) to 1132 bp (ORF and 3’ UTR). B: The <i>SIX1</i> gene, <i>hph</i> gene and <i>Neo</i> gene fragments were amplified by PCR to confirm the correct complementation mutants. C: Disease symptoms of cabbage seedlings inoculated with wild type isolate Foc, deletion mutants Foc-ΔSIX1, and complementation mutants Foc-ΔSIX1::Foc-SIX1 were shown at 14 dpi. Similar results were obtained in three independent biological replicates.</p

Construction and verification of deletion mutants Foc-ΔSIX1.

<p>A: <i>Foc-SIX1</i> gene deletion and replacement with an intact selectable marker gene (<i>hph</i>) by homologous recombination. B: Confirmation of the correct deletion by PCR. Numbers 1, 2, 3 and 4 represent the four primer pairs that specify the <i>SIX1</i> gene, <i>hph</i> gene, and the correct upstream and downstream homologous recombination, respectively. C: Southern hybridization analysis of wild type isolate and mutants Foc-ΔSIX1. The genomic DNAs from the wild type Foc isolate and deletion mutants Foc-ΔSIX1 were digested with <i>Sal</i> I. A fragment amplified from upstream of the target gene was used as probe.</p

Construction, verification, and virulence analysis of transformants Foc-ΔSIX1::Fol-SIX1.

<p>A: Foc-ΔSIX1 mutants were complemented by transformation with approximately 2.0 kb of <i>Fol-SIX1</i> spanning from -832 bp (promoter relative to start codon) to 1091 bp (ORF and 3’ UTR). B: The <i>Fol-SIX1</i> gene and <i>Neo</i> gene fragments were amplified by PCR to confirm the correct complementation mutants. C: Disease symptoms of cabbage seedlings inoculated with wild type isolate Foc, deletion mutants Foc-ΔSIX1 (D1 and D2), complementation mutants (C2 and C3) and transformants Foc-ΔSIX1::Fol-SIX1 (T1 and T2) were shown at 18 dpi. Similar results were obtained in three independent biological replicates.</p

A SIX1 Homolog in <i>Fusarium oxysporum</i> f. sp. <i>conglutinans</i> Is Required for Full Virulence on Cabbage

<div><p><i>Fusarium oxysporum</i> is a soil-born fungus that induces wilt and root rot on a variety of plants. <i>F</i>. <i>oxysporum</i> f. sp. <i>conglutinans</i> (Foc) can cause wilt disease on cabbage. This study showed that a homolog of SIX1 protein in the <i>Arabidopsis</i> infecting isolate Fo5176 (Fo5176-SIX1) had four isoforms in the conidia of Foc by proteomic analysis. Thus, we analyzed the roles of protein Foc-SIX1. Gene expression analysis showed that, compared to the expression in mycelia, dramatically altered expression of <i>Foc-SIX1</i> could be detected after infecting cabbages, and <i>Foc-SIX1</i> was highly expressed in conidia under axenic culture condition. Furthermore, we knocked out the <i>Foc-SIX1</i> gene and found that Foc-ΔSIX1 mutants had significantly reduced virulence compared with wild type isolate, and full virulence was restored by complementation of Foc-ΔSIX1 mutants with <i>Foc-SIX1</i>. Thus, we concluded that SIX1 in Foc was required for full virulence on cabbage. We also complemented Foc-ΔSIX1 with <i>SIX1</i> gene in <i>F</i>. <i>oxysporum</i> f. sp. <i>lycopersici</i> (Fol) and found Foc-ΔSIX1::Fol-SIX1 mutants did not affect the virulence of Foc-ΔSIX1. The results confirmed that Fol-SIX1 was not capable of replacing the role of Foc-SIX1 in Foc on the disease symptom development of cabbage. The roles of Fol-SIX1 on virulence might rely on host specificity.</p></div

Altered Resting-State Brain Activity and Connectivity in Depressed Parkinson’s Disease

<div><p>Depressive symptoms are common in Parkinson’s disease (PD), but the neurophysiological mechanisms of depression in PD are poorly understood. The current study attempted to examine disrupted spontaneous local brain activities and functional connectivities that underlie the depression in PD. We recruited a total of 20 depressed PD patients (DPD), 40 non-depressed PD patients (NDPD) and 43 matched healthy controls (HC). All the subjects underwent neuropsychological tests and resting-state fMRI scanning. The between-group differences in the amplitude of low frequency fluctuations (ALFF) of BOLD signals were examined using post-hoc tests after the analysis of covariance. Compared with the NDPD and HC, the DPD group showed significantly increased ALFF in the left median cingulated cortex (MCC). The functional connectivity (FC) between left MCC and all the other voxels in the brain were then calculated. Compared with the HC and NDPD group, the DPD patients showed stronger FC between the left MCC and some of the major nodes of the default mode network (DMN), including the post cingulated cortex/precuneus, medial prefrontal cortex, inferior frontal gyrus, and cerebellum. Correlation analysis revealed that both the ALFF values in the left MCC and the FC between the left MCC and the nodes of DMN were significantly correlated with the Hamilton Depression Rating Scale score. Moreover, higher local activities in the left MCC were associated with increased functional connections between the MCC and the nodes of DMN in PD. These abnormal activities and connectivities of the limbic-cortical circuit may indicate impaired high-order cortical control or uncontrol of negative mood in DPD, which suggested a possible neural mechanism of the depression in PD.</p></div

Statistical parametric map showing the significant differences in the ALFF between three groups: DPD, NDPD and HC.

<p>The ANCOVA thresholds were set at a voxel-level <i>p</i> < 0.01, cluster size > 918 mm³ /34 voxels, corresponding to a corrected <i>p</i> < 0.05 as determined by AlphaSim correction. A two-sample post hoc <i>t</i>-test was performed between each pair of the three groups (DPD vs NDPD, DPD vs HC, NDPD vs HC)(voxel-level <i>p</i> < 0.01, cluster size > 162 mm³ /6 voxels, corresponding to a corrected <i>p</i> < 0.05 as determined by AlphaSim correction). Statistical parametric map showing the significant correlations between HAMD score and ALFF value in left MCC.</p