RNA interference (RNAi) is a technique used for transgene-mediated gene silencing based on the mechanism of posttranscriptional gene silencing (PTGS). PTGS is an ubiquitous basic biological phenomenon involved in the regulation of transcript abundance and plants’ immune response to viruses. PTGS also mediates genomic stability by silencing of retroelements. RNAi has become an important research tool for studying gene function by strong and selective suppression of target genes. Here, we present si-Fi, a software tool for design optimization of RNAi constructs necessary for specific target gene knock-down. It offers efficiency prediction of RNAi sequences and off-target search, required for the practical application of RNAi. si-Fi is an open-source (CC BY-SA license) desktop software that works in Microsoft Windows environment and can use custom sequence databases in standard FASTA format.

Significance: Holocentric chromosomes are characterized by kinetochore activity along each sister chromatid. Although the kinetochore structure seems to be well conserved, as in monocentric organisms, the organization of holocentromeres is still elusive, and no centromeric repeat has been found associated with centromeric histone H3 variant-positive centromeric nucleosomes for any holocentric organism studied hitherto. We demonstrate that holocentrics of the sedge (Cyperaceae)Rhynchospora puberapossess different classes of centromere-specific repeats. Holocentromeres are composed of multiple centromeric units interspersing the gene-containing chromatin, and, as a functional adaption, a cell-cycle–dependent shuffling of centromeric units results in the formation of functional (poly)centromeres during cell division. The genome-wide distribution of centromeric repeat arrays interspersing the euchromatin provides a previously unidentified type of centromere organization.

The rapid increase in heat shock proteins upon exposure to damaging stresses and during plant development related to desiccation events reveal their dual importance in plant development and stress tolerance. Genome-wide sequence survey identified 20 non-redundant small heat shock proteins (sHsp) and 22 heat shock factor (Hsf) genes in barley. While all three major classes (A, B, C) of Hsfs are localized in nucleus, the 20 sHsp gene family members are localized in different cell organelles like cytoplasm, mitochondria, plastid and peroxisomes. Hsf and sHsp members are differentially regulated during drought and at different seed developmental stages suggesting the importance of chaperone role under drought as well as seed development. In silico cis-regulatory motif analysis of Hsf promoters showed an enrichment with abscisic acid responsive cis-elements (ABRE), implying regulatory role of ABA in mediating transcriptional response of HvsHsf genes. Gene regulatory network analysis identified HvHsfB2c as potential central regulator of the seed-specific expression of several HvsHsps including 17.5CI sHsp. These results indicate that HvHsfB2c is co-expressed in the central hub of small Hsps and therefore it may be regulating the expression of several HvsHsp subclasses HvHsp16.88-CI, HvHsp17.5-CI and HvHsp17.7-CI. The in vivo relevance of binding specificity of HvHsfB2C transcription factor to HSE-element present in the promoter of HvSHP17.5-CI under heat stress exposure is confirmed by gel shift and LUC-reporter assays. Further, we isolated 477 bp cDNA from barley encoding a 17.5 sHsp polypeptide, which was predominantly upregulated under drought stress treatments and also preferentially expressed in developing seeds. Recombinant HvsHsp17.5-CI protein was expressed in E. coli and purified to homogeneity, which displayed in vitro chaperone activity. The predicted structural model of HvsHsp-17.5-CI protein suggests that the α-crystallin domain is evolutionarily highly conserved.

Crop plants are regularly challenged by a range of environmental stresses which typically retard their growth and ultimately compromise economic yield. The stress response involves the reprogramming of approximately 4% of the transcriptome. Here, the behavior of AtRD22 and AtUSPL1, both members of the Arabidopsis thaliana BURP (BNM2, USP, RD22 and polygalacturonase isozyme) domain-containing gene family, has been characterized. Both genes are up-regulated as part of the abscisic acid (ABA) mediated moisture stress response. While AtRD22 transcript was largely restricted to the leaf, that of AtUSPL1 was more prevalent in the root. As the loss of function of either gene increased the plant's moisture stress tolerance, the implication was that their products act to suppress the drought stress response. In addition to the known involvement of AtUSPL1 in seed development, a further role in stress tolerance was demonstrated. Based on transcriptomic data and phenotype we concluded that the enhanced moisture stress tolerance of the two loss-of-function mutants is a consequence of an enhanced basal defense response.

We have identified a DNA methyltransferase of the Dnmt2 family in Dictyostelium that was denominated DnmA. Expression of the dnmA gene is downregulated during the developmental cycle. Overall DNA methylation in Dictyostelium is approximately 0.2% of the cytosine residues, which indicates its restriction to a limited set of genomic loci. Bisulfite sequencing of specific sites revealed that DnmA is responsible for methylation of mostly asymmetric C-residues in the retrotransposons DIRS-1 and Skipper. Disruption of the gene resulted in a loss of methylation and in increased transcription and mobilization of Skipper. Skipper transcription was also upregulated in strains that had genes encoding components of the RNA interference pathway disrupted. In contrast, DIRS-1 expression was not affected by a loss of DnmA but was strongly increased in strains that had the RNA-directed RNA polymerase gene rrpC disrupted. A large number of siRNAs were found that corresponded to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA modification. No siRNAs corresponding to the standard Skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene silencing in Dictyostelium but that different, partially overlapping mechanisms control transposon silencing.

The centromeric histone H3 variant cenH3 is an essential centromeric protein required for assembly, maintenance, and proper function of kinetochores during mitosis and meiosis. We identified a kinetochore null2 (KNL2) homolog in Arabidopsis thaliana and uncovered features of its role in cenH3 loading at centromeres. We show that Arabidopsis KNL2 colocalizes with cenH3 and is associated with centromeres during all stages of the mitotic cell cycle, except from metaphase to mid-anaphase. KNL2 is regulated by the proteasome degradation pathway. The KNL2 promoter is mainly active in meristematic tissues, similar to the cenH3 promoter. A knockout mutant for KNL2 shows a reduced level of cenH3 expression and reduced amount of cenH3 protein at chromocenters of meristematic nuclei, anaphase bridges during mitosis, micronuclei in pollen tetrads, and 30% seed abortion. Moreover, knl2 mutant plants display reduced expression of suppressor of variegation 3-9 homologs2, 4, and 9 and reduced DNA methylation, suggesting an impact of KNL2 on the epigenetic environment for centromere maintenance.