Daolong Dou

Island Cotton Gbve1 Gene Encoding A Receptor-Like Protein Confers Resistance to Both Defoliating and Non-Defoliating Isolates of Verticillium dahliae

Verticillium wilt caused by soilborne fungus Verticillium dahliae could significantly reduce cotton yield. Here, we cloned a tomato Ve homologous gene, Gbve1, from an island cotton cultivar that is resistant to Verticillium wilt. We found that the Gbve1 gene was induced by V. dahliae and by phytohormones salicylic acid, jasmonic acid, and ethylene, but not by abscisic acid. The induction of Gbve1 in resistant cotton was quicker and stronger than in Verticillium-susceptible upland cotton following V. dahliae inoculation. Gbve1 promoter-driving GUS activity was found exclusively in the vascular bundles of roots and stems of transgenic Arabidopsis. Virus-induced silencing of endogenous genes in resistant cotton via targeting a fragment of the Gbve1 gene compromised cotton resistance to V. dahliae. Furthermore, we transformed the Gbve1 gene into Arabidopsis and upland cotton through Agrobacterium-mediated transformation. Overexpression of the Gbve1 gene endowed transgenic Arabidopsis and upland cotton with resistance to high aggressive defoliating and non-defoliating isolates of V. dahliae. And HR-mimic cell death was observed in the transgenic Arabidopsis. Our results demonstrate that the Gbve1 gene is responsible for resistance to V. dahliae in island cotton and can be used for breeding cotton varieties that are resistant to Verticillium wilt.

Microarray profiling reveals microRNAs involving soybean resistance to Phytophthora sojae

MicroRNAs (miRNAs), a group of small noncoding RNAs, may serve as a class of post-transcriptional regulators in plant immune systems. Nevertheless, little is known about their roles in plant immune response to the oomycete pathogens. To identify miRNAs involved in the response of soybean to Phytophthora sojae, we examined expressional patterns of miRNAs upon infection by P. sojae by microarray analysis in three soybean cultivars: Williams (susceptible), Conrad (quantitative resistance), and Williams 82 (qualitative resistance). Expression of a number of miRNAs was significantly altered upon infection and (or) in the different genotypes. qRT-PCR data with some miRNAs further confirmed the microarray results. Comparative analysis of the selected miRNAs and their targeted gene expression datasets uncovered many reciprocally expressed miRNA-target pairs, which could proposed a feedback circuit between miRNA(s) and protein-coding genes. These results may serve as a basis for further in-depth studies of miRNAs involved in soybean resistance to P. sojae.

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.

Phytophthora sojae Effector PsCRN70 Suppresses Plant Defenses in Nicotiana benthamiana

Phytophthora sojae, an oomycete pathogen, produces a large number of effector proteins that enter into host cells. The Crinklers (Crinkling and Necrosis, CRN) are cytoplasmic effectors that are conserved in oomycete pathogens and their encoding genes are highly expressed at the infective stages in P. sojae. However, their roles in pathogenesis are largely unknown. Here, we functionally characterized an effector PsCRN70 by transiently and stably overexpressing it in Nicotiana benthamiana. We demonstrated that PsCRN70 was localized to the plant cell nucleus and suppressed cell death elicited by all the tested cell death-inducing proteins, including BAX, PsAvh241, PsCRN63, PsojNIP and R3a/Avr3a. Overexpression of the PsCRN70 gene in N. benthamiana enhanced susceptibility to P. parasitica. The H2O2 accumulation in the PsCRN70-transgenic plants was reduced compared to the GFP-lines. The transcriptional levels of the defense-associated genes, including PR1b, PR2b, ERF1 and LOX, were also down-regulated in the PsCRN70-transgenic lines. Our results suggest that PsCRN70 may function as a universal suppressor of the cell death induced by many elicitors, the host H2O2 accumulation and the expression of defense-associated genes, and therefore promotes pathogen infection.

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 Phytophthora sojae 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 Nicotiana benthamiana 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.

A Virulence Essential CRN Effector of Phytophthora capsici Suppresses Host Defense and Induces Cell Death in Plant Nucleus

Phytophthora capsici is a soil-borne plant pathogen with a wide range of hosts. The pathogen secretes a large array of effectors during infection of host plants, including Crinkler (CRN) effectors. However, it remains largely unknown on the roles of these effectors in virulence especially in P. capsici. In this study, we identified a cell death-inducing CRN effector PcCRN4 using agroinfiltration approach. Transient expression of PcCRN4 gene induced cell death in N. benthamiana, N. tabacum and Solanum lycopersicum. Overexpression of the gene in N. benthamiana enhanced susceptibility to P. capsici. Subcellular localization results showed that PcCRN4 localized to the plant nucleus, and the localization was required for both of its cell death-inducing activity and virulent function. Silencing PcCRN4 gene in P. capsici significantly reduced pathogen virulence. The expression of the pathogenesis-related gene PR1b in N. benthamiana was significantly induced when plants were inoculated with PcCRN4-silenced P. capsici transformant compared to the wilt-type. Callose deposits were also abundant at sites inoculated with PcCRN4-silenced transformant, indicating that silencing of PcCRN4 in P. capsici reduced the ability of the pathogen to suppress plant defenses. Transcriptions of cell death-related genes were affected when PcCRN4-silenced line were inoculated on Arabidopsis thaliana, suggesting that PcCRN4 may induce cell death by manipulating cell death-related genes. Overall, our results demonstrate that PcCRN4 is a virulence essential effector and it needs target to the plant nucleus to suppress plant immune responses.

GmCYP82A3, a Soybean Cytochrome P450 Family Gene Involved in the Jasmonic Acid and Ethylene Signaling Pathway, Enhances Plant Resistance to Biotic and Abiotic Stresses

The cytochrome P450 monooxygenases (P450s) represent a large and important enzyme superfamily in plants. They catalyze numerous monooxygenation/hydroxylation reactions in biochemical pathways, P450s are involved in a variety of metabolic pathways and participate in the homeostasis of phytohormones. The CYP82 family genes specifically reside in dicots and are usually induced by distinct environmental stresses. However, their functions are largely unknown, especially in soybean (Glycine max L.). Here, we report the function of GmCYP82A3, a gene from soybean CYP82 family. Its expression was induced by Phytophthora sojae infection, salinity and drought stresses, and treatment with methyl jasmonate (MeJA) or ethephon (ETH). Its expression levels were consistently high in resistant cultivars. Transgenic Nicotiana benthamiana plants overexpressing GmCYP82A3 exhibited strong resistance to Botrytis cinerea and Phytophthora parasitica, and enhanced tolerance to salinity and drought stresses. Furthermore, transgenic plants were less sensitive to jasmonic acid (JA), and the enhanced resistance was accompanied with increased expression of the JA/ET signaling pathway-related genes.

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.

Phytophthora sojae TatD Nuclease Positively Regulates Sporulation and Negatively Regulates Pathogenesis

During pathogenic interactions, both the host and pathogen are exposed to conditions that induce programmed cell death (PCD). Certain aspects of PCD have been recently examined in eukaryotic microbes but not in oomycetes. Here, we identified conserved TatD proteins in Phytophthora sojae; the proteins are key components of DNA degradation in apoptosis. We selected PsTatD4 for further investigation because the enzyme is unique to the oomycete branch of the phylogenetic tree. The purified protein exhibited DNase activity in vitro. Its expression was upregulated in sporangia and later infective stages but downregulated in cysts and during early infection. Functional analysis revealed that the gene was required for sporulation and zoospore production, and the expression levels were associated with the numbers of hydrogen-peroxide-induced terminal dUTP nick end-labeling-positive cells. Furthermore, overexpression of PsTatD4 gene reduced the virulence in a susceptible soybean cultivar. Together, these data suggest that apoptosis may play different roles in the early and late infective stages of P. sojae, and that PsTatD4 is a key regulator of infection. The association of PsTatD4 and apoptosis will lay a foundation to understanding the basic biology of apoptosis and its roles in P. sojae disease cycle.