The genome assembly and annotation of<i> </i><i>Puccinia striiformis</i> f. sp.<i> tritici</i> isolate AZ2

PacBio HiFi sequencing technology and a trio-binning approach were combined to generate a fully haplotype-resolved and nearly gap-free genome assembly of Puccinia striiformis f. sp. tritici (Pst) isolate AZ2, which was derived from Pst isolate A153 crossing with isolate XZ-2. AZ2A and AZ2B represent the two haplotypes of AZ2. AZ2mt represents the mitochondrial genome of AZ2.</p

Microarray-based identification of conserved microRNA from wheat and their expression profiles response to <i>Puccinia striiformis</i> f. sp. <i>tritici</i>

<div><p></p><p>MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level and play a critical role in many important biological processes of plants. Wheat stripe rust is one of the most destructive fungal diseases of wheat worldwide, yet the roles of wheat miRNAs in response to <i>Puccinia striiformis</i> f. sp. <i>tritici</i> (<i>Pst</i>) are largely unknown. Here, we report a simple array platform that could detect 188 plant miRNAs in 95 miRNAs families from eight plant species. We identified two new members of conserved miRNAs families and five known miRNAs using the platform and RNA gel blot analysis. The transcript accumulation of seven miRNAs was detected in wheat leaves ‘Suwon 11’ inoculated with <i>Pst</i> using stem-loop real-time quantitative PCR (RT-qPCR). By analysing their predicted target genes, we discuss and propose the basal roles for miRNAs in the interaction between wheat and <i>Pst</i>, with most of the target genes being stress-related.</p></div

Cluster and heat map of DEGs of XZ at the adult plant stage.

<p>The heat map shows the gene expression obtained by the clustering affinity search technique. Each line refers to the data for one gene. The color bar represents the log2 of fold change values and ranges from green (−8) to red (8). The six DGE libraries included non-inoculated adult plants at 24 hours post-inoculation (hpi) (Ak-M-24), 48 hpi (Ak-M-48) and 120 hpi (Ak-M-120), and inoculated adult plants at 24 hpi (Ak-I-24), 48 hpi (Ak-I-48) and 120 hpi (Ak-I-120). A: The DEGs that demonstrated at least a two-fold difference in each of the three comparisons at the adult plant stage. B: The up-regulated unigenes that demonstrated at least a four-fold change in each of the three comparisons at the adult plant stage.</p

Quantitative real-time polymerase chain reaction analysis of the relative transcript levels of the six candidate unigenes induced by <i>Pst</i> infection at seedling and adult stages.

<p>The relative expression levels of the unigenes were calculated by the comparative threshold method (2^–ΔΔCT) and were relative to that at the 0 hour time point. The results are presented as the means ± standard errors of three biological replications. The different letters represent significant differences [<i>P</i>≤0.05 according to analysis of variance (ANOVA)].</p

Statistical chart of DEGs during <i>Pst</i> infection at the adult plant stage.

<p>Differentially expressed genes were identified by filtering the two-fold up-regulated and down-regulated genes with FDR≤10⁻⁴. The bars represent the number of up-regulated (black) and down-regulated (gray) unigenes. The six DGE libraries included non-inoculated adult plants at 24 hours post-inoculation (hpi) (Ak-M-24), 48 hpi (Ak-M-48) and 120 hpi (Ak-M-120), and inoculated adult plants at 24 hpi (Ak-I-24), 48 hpi (Ak-I-48) and 120 hpi (Ak-I-120).</p

Functional analysis of six candidate genes during the interaction between XZ and stripe rust using the BSMV-mediated virus-induced gene silencing system.

<p>The second top leaves were pre-inoculated with BSMV:γ or recombinant BSMV followed by inoculation with <i>Puccinia striiformis</i> f. sp. <i>tritici</i> race CYR32. (A) Necrotic area, the average area was calculated by DP-BSW software. wheat37392_refgene, wheat12902_refgene, wheat75137_refgene or wheat31306_refgene -silenced plants showed a significant decrease at 120 hours post-inoculation (hpi). (B) Hyphal length, the length of IH was measured from the substomatal vesicle to the apex of the longest infection hyphae, Wheat-37392_refgene, wheat12902_refgene, wheat75137_refgene and wheat31306_refgene -silenced plants showed a significant increase at 120 hpi. (C) Wheat37392_refgene-silenced plants showed a significant decrease in the content of lignin at 48 and 120 hpi. (D) Wheat- 37392_refgene or 12902_refgene -silenced plants showed a decrease in the content of SA at 48 and 120 hpi, but no significant decrease between control and silenced plants. (E) Significant decrease in ROS accumulation in wheat-12902_refgene, wheat-75137_refgene and wheat-31306_refgene -silenced plants at 120 hpi. (F) No difference in the content of Chloroplast was observed between control and wheat-36302_refgene and wheat 12266_refgene -silenced plants at 120 hpi. The error bars represent the variations among three independent replicates. The different letters represent significant differences [<i>P</i>≤0.05 according to analysis of variance (ANOVA)].</p

Verification of DGE-Seq results by qRT-PCR.

<p>Verification of DGE-Seq results by qRT-PCR.</p

Relative transcript levels of candidate genes in candidate gene-knockdown wheat leaves.

<p>RNA samples were isolated from the flag leaves of wheat infected with BSMV:γ, BSMV:wheat37392_refgene, BSMV:wheat12902_refgene, BSMV:wheat75137_refgene, BSMV:wheat31306_refgene, BSMV:wheat36302_refgene and BSMV:wheat12266_refgene at 0, 24, 48 and 120 hours post-inoculation (hpi) with <i>Pst</i> CYR32. The error bars represent the variations among three independent replicates. The different letters represent significant differences [<i>P</i>≤0.05 according to analysis of variance (ANOVA)]. The relative gene expression levels were quantified using the comparative threshold (2^-ΔΔCT) method and compared with that of BSMV:γ.</p

Statistical chart of enrichments in ‘biological processes’ during <i>Pst</i> infection at the adult plant stage.

<p>The leaves of adult plants were inoculated with <i>Pst</i> CYR32. The panels represent the transcriptional changes in ‘biological processes’ obtained from the Ak-I-24 vs. Ak-M-24, Ak-I-48 vs. Ak-M-48 and Ak-I-120 vs. Ak-M-120 comparisons. The six DGE libraries included non-inoculated adult plants at 24 hours post-inoculation (hpi) (Ak-M-24), 48 hpi (Ak-M-48) and 120 hpi (Ak-M-120), and inoculated adult plants at 24 hpi (Ak-I-24), 48 hpi (Ak-I-48) and 120 hpi (Ak-I-120).</p

Output statistics from sequencing.

<p>Output statistics from sequencing.</p