Computational identification of novel microRNAs and targets in Glycine max.

Plant miRNAs, the endogenous non-coding small RNAs of about 20-24 nucleotides, play important roles in multiple biological processes by acting as negative regulators of their targeted mRNAs. Soybean (Glycine max (L.) Merr.) is one of the important oil crops of the world, in which many miRNAs have been obtained through the computational prediction or experiments. However, the miRNA genes identified for soybean are still far from saturation, and their biological functions are largely unknown. Here, a total of 48 candidates of miRNAs were identified following a range of strict filtering criteria. Detailed sequence analysis showed that G. max pre-miRNAs vary in length from 47 to 380 nt, embody mature miRNAs that differ in their physical location within the pre-miRNAs. In this study, twenty miRNAs were confirmed by microarray and three miRNAs were further validated by poly(A)-tailed RT-PCR. Comparative sequence analysis of soybean miRNA sequences showed that uracil is the dominant base in the first position at the 5' end of the mature miRNAs, and the base may have an important functional role in miRNA biogenesis and/or miRNA-mediated gene regulation. Finally, we predicted potential targets of these miRNAs. These target genes were predicted to encode transcription factors, resistance protein, heat shock protein, protein kinase, transporter, zinc finger protein and others, which might play important roles in soybean development and stress response.

Two RxLR avirulence genes in Phytophthora sojae determine soybean Rps1k-mediated disease resistance.

Resistance to Phytophthora sojae (Rps) genes have been widely used in soybean against root and stem rot diseases caused by this oomycete. Among 15 known soybean Rps genes, Rps1k has been the most widely used in the past four decades. Here, we show that the products of two distinct but closely linked RxLR effector genes are detected by Rps1k-containing plants, resulting in disease resistance. One of the genes is Avr1b-1, that confers avirulence in the presence of Rps1b. Three lines of evidence, including overexpression and gene silencing of Avr1b-1 in stable P. sojae transformants, as well as transient expression of this gene in soybean, indicated that Avr1b could trigger an Rps1k-mediated defense response. Some isolates of P. sojae that do not express Avr1b are nevertheless unable to infect Rps1k plants. In those isolates, we identified a second RxLR effector gene (designated Avr1k), located 5 kb away from Avr1b-1. Silencing or overexpression of Avr1k in P. sojae stable transformants resulted in the loss or gain, respectively, of the avirulence phenotype in the presence of Rps1k. Only isolates of P. sojae with mutant alleles of both Avr1b-1 and Avr1k could evade perception by the soybean plants carrying Rps1k.

Characterization of genipin-crosslinked gelatin/hyaluronic acid-based hydrogel membranes and loaded with hinokitiol: In vitro evaluation of antibacterial activity and biocompatibility.

Hydrogel membranes are often used as physical barriers in oral tissue reconstruction and facial surgery to isolate connective and epithelial tissues and form a closed space for undisturbed bone healing. In this study, gelatin and hyaluronic acid were crosslinked with genipin and loaded with a hinokitiol additive as a bacteriostatic agent for potential applications as regeneration membranes. This bifunctional membrane had biocompatibility and antibacterial activities on each membrane side for proper biodegradation. Different membrane groups of gelatin/hyaluronic acid were obtained via a solution casting technique and were genipin crosslinked. The membrane groups were further loaded with adequate hinokitiol at a loading concentration of up to 0.16 g/L (hinokitiol to phosphate buffered saline). Fourier transform infrared spectroscopy showed that gelatin and hyaluronic acid were crosslinked with genipin through cross-linking amide bond (CONH) formation with a cross-linking degree of over 84%. The groups with hinokitiol showed substantial antibacterial activity. Meanwhile, the addition of hinokitiol on hydrogel membranes did not significantly affect the tensile strength. However, it decreased the solubility of the membranes by slowing down the relaxation and degradation of their molecular junctions as hinokitiol is a hydrophobic compound with low permeability. Consequently, the degradation of hydrogel membranes with hinokitiol was delayed. In vitro cytocompatibility indicated that the cell viability of the groups with hinokitiol increased with incubation time, demonstrating that cell viability and proliferation were not affected by cell culture testing.

Identification and functional characterization of the soybean GmaPPO12 promoter conferring Phytophthora sojae induced expression.

Identification of pathogen-inducible promoters largely lags behind cloning of the genes for disease resistance. Here, we cloned the soybean GmaPPO12 gene and found that it was rapidly and strongly induced by Phytophthorasojae infection. Computational analysis revealed that its promoter contained many known cis-elements, including several defense related transcriptional factor-binding boxes. We showed that the promoter could mediate induction of GUS expression upon infection in both transient expression assays in Nicotianabenthamiana and stable transgenic soybean hairy roots. Importantly, we demonstrated that pathogen-induced expression of the GmaPPO12 promoter was higher than that of the soybean GmaPR1a promoter. A progressive 5' and 3' deletion analysis revealed two fragments that were essential for promoter activity. Thus, the cloned promoter could be used in transgenic plants to enhance resistance to phytophthora pathogens, and the identified fragment could serve as a candidate to produce synthetic pathogen-induced promoters.

Intracellular and extracellular phosphatidylinositol 3-phosphate produced by Phytophthora species is important for infection.

RxLR effectors produced by Phytophthora pathogens have been proposed to bind to phosphatidylinositol 3-phosphate (PtdIns(3)P) to mediate their translocation into host cells and/or to increase their stability in planta. Since the levels of PtdIns(3)P in plants are low, we examined whether Phytophthora species may produce PtdIns(3)P to promote infection. We observed that PtdIns(3)P-specific GFP biosensors could bind to P. parasitica and P. sojae hyphae during infection of Nicotiana benthamiana leaves transiently secreting the biosensors, suggesting that the hyphae exposed PtdIns(3)P on their plasma membrane and/or secreted PtdIns(3)P. Silencing of the phosphatidylinositol 3-kinases (PI3K) genes, treatment with LY294002, or expression of PtdIns(3)P-binding proteins by P. sojae reduced the virulence of the pathogen on soybean, indicating that pathogen-synthesized PtdIns(3)P was required for full virulence. Secretion of PtdIns(3)P-binding proteins or of a PI3P-5-kinase by N. benthamiana leaves significantly increased the level of resistance to infection by P. parasitica and P. capsici. Together, our results support the hypothesis that Phytophthora species produce external PtdIns(3)P to aid in infection, such as to promote entry of RxLR effectors into host cells. Our results derived from P. sojae RxLR effector Avr1b confirm that both the N-terminus and the C-terminus of this effector can bind PtdIns(3)P.

Gene duplication and fragment recombination drive functional diversification of a superfamily of cytoplasmic effectors in Phytophthora sojae.

Phytophthora and other oomycetes secrete a large number of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling and necrosis inducing proteins (CRN), or Crinkler. Here, we first investigated the evolutionary patterns and mechanisms of CRN effectors in Phytophthora sojae and compared them to two other Phytophthora species. The genes encoding CRN effectors could be divided into 45 orthologous gene groups (OGG), and most OGGs unequally distributed in the three species, in which each underwent large number of gene gains or losses, indicating that the CRN genes expanded after species evolution in Phytophthora and evolved through pathoadaptation. The 134 expanded genes in P. sojae encoded family proteins including 82 functional genes and expressed at higher levels while the other 68 genes encoding orphan proteins were less expressed and contained 50 pseudogenes. Furthermore, we demonstrated that most expanded genes underwent gene duplication or/and fragment recombination. Three different mechanisms that drove gene duplication or recombination were identified. Finally, the expanded CRN effectors exhibited varying pathogenic functions, including induction of programmed cell death (PCD) and suppression of PCD through PAMP-triggered immunity or/and effector-triggered immunity. Overall, these results suggest that gene duplication and fragment recombination may be two mechanisms that drive the expansion and neofunctionalization of the CRN family in P. sojae, which aids in understanding the roles of CRN effectors within each oomycete pathogen.