GmSKP1,a Novel S-phase Kinase-associated Protein 1 in Glycine max,Enhancing Resistance Against Phytophthora sojae Infection

Phytophthora root and stem rot of soybean caused by Phytophthora sojae(P.sojae)is a devastating disease that affects soybean[Glycine max(L.)Merr.]all over the world.S-phase kinase-associated protein 1(SKP1)proteins are key members of the SKP1/Cullin/F-box protein(SCF)ubiquitin ligase complex and play diverse roles in plant biology.However,the role of SKP1 in soybean against the phytopathogenic oomycete P.sojae remains unclear.In this study,a novel member of the soybean SKP1 gene family,GmSKP1 which was significantly induced by P.sojae,was reported.The expression of GmSKP1 was simultaneously induced by methyl jasmonate(MeJA),salicylic acid(SA)and ethylene(ET),which might suggest an important role for GmSKP1 of plant in responses to hormone treatments.Functional analysis using GmSKP1 overexpression lines showed that GmSKP1 enhanced resistance to P.sojae in transgenic soybean plants.Further analyses showed that GmSKP1 interacted with a homeodomain-leucine zipper protein transcription factor(GmHDL56)and a WRKY transcription factor(GmWRKY31),which could positively regulate responses to P.sojae in soybean.Importantly,several pathogenesis-related(PR)genes were constitutively activated,including GmPR1a,GmPR2,GmPR3,GmPR4,GmPR5a and GmPR10,in GmSKP1-OE soybean plants.Taken together,these results suggested that GmSKP1 enhanced resistance to P.sojae in soybean,possibly by activating the defense-related PR genes.

Phylogenetic Analysis of the Sequences of rDNA Internal Transcribed Spacer (ITS) of Phytophthora sojae

The internal transcribed spacer （ITS） region （ITS1, ITS2 and 5.8S rDNA） of the nuclear ribosomal DNA （nrDNA） was amplified via the PCR method in seventeen different isolates of Phytophthora sojae using the common primers of the ITS of fungi. Around 800 bp- 1,000 bp fragments were obtained based on the DL2000 marker and the sequences of the PCR products were tested. Taking isolate USA as outgroup, the phylogenetic tree was constructed by means of maximum parsimony analysis, and the genetic evolution among isolates was analyzed. The results showed that there is a great difference between the base constitution of ITS 1 and ITS2 among various isolates. The seventeen isolates are classified into three groups, and the isolates from the same region belong to the same group, which shows the variation in geography.

Screening and Identification of Antagonistic Strain against Phytophthora sojae

[ Objective] The paper was to screen the antagonistic strain against Phytophthora sojae with biocontrol potential, and provide basis for searching control measures and designing new control strategies against P. sojae. [ Method] The rhizosphere soil of soybean was collected from three different places in Heilongjiang Province, and various soil microorganisms were isolated. Dual culture method was used to screen the microorganism with antagonistic effect against P. sojae. On this basis, the growth inhibition rate of the microorganism with stronger antagonistic effect against P. sojae was determined, and its control effect against P. sojae was also measured. [ Result] A strain of bacterium with relatively good antagonistic effect was isolated from soil, and named as strain B048. Dual test showed that the growth inhibition rate of antagonistic bacterium 11048 against P. sojac reached 97.5%. Antagonistic endurance tests showed that the width of inhibition zone was still 20.0 mm after dual culture with P. sojac for21 d. In potting experiment, the control effect of B048 against P. sojae was 100%. The antagonistic bacterium was primarily identified to be Bacillus pumilus through morphology and 16S rDNA sequence analysis. [Condusion] The antagonistic bacterium B048 had good prospect to be developed as the biocontrol bacterium against P. sojae.

Identification, Genetic Analysis and Mapping of Resistance to Phytophthora sojae of Pm28 in Soybean

Phytophthora sojae Kanfman and Gerdemann （P. sojae） is one of the most prevalent pathogens and causes Phytophthora root rot, which limits soybean production worldwide. Development of resistant cultivars is a cost-effective approach to controlling this disease. In this study, 127 soybean germplasm were evaluated for their responses to Phytophthora sojae strain Pm28 using the hypocotyl inoculation technique, and 49 were found resistant to the strain. The hypocotyl of P1, P2, F1, and F2：3 of two crosses of Ludou 4 （resistant）×Youchu 4 （susceptible） and Cangdou 5 （resistant）×Williams （susceptible） were inoculated with Pm28, and were used to analyze the inheritance of resistance. The population derived from the cross of Ludou 4×Youchu 4 was used to map the resistance gene （designated as Rps9） to a linkage group. 932 pairs of SSR primers were used to detect polymorphism, and seven SSR markers were mapped near the resistance gene. The results showed that the resistance to Pm28 in Ludou 4 and Cangdou 5 was controlled by a single dominant gene Rps9, which was located on the molecular linkage group N between the SSR markers Satt631 （7.5 cM） and Sat_186 （4.3 cM）.

Distribution and Virulence Diversity of Phytophthora sojae in China

By investigating occurrence of Phytophthora root rot in fields and isolating P.sojae fromdiseased plants and soils, the distribution of P.sojae in China was surveyed. In addition tonortheast region, P.sojae existed in Huanghe-Huaihe basin and Yangtze basin too. Eighty- threeisolates of P.sojae isolated from different areas were identified on virulence using 13differential soybean cultivars, abundant virulence diversity was found in P.sojae. The greaterdiversity in virulence of P.sojae was in isolates from soil than from plants. And the greatestvirulence diversity of P.sojae was found in Yangtze basin.

Differential Gene and Protein Expression in Soybean at Early Stages of Incompatible Interaction with Phytophthora sojae

Soybean root and stem rot caused by Phytophthora sojae is a destructive disease worldwide. Using genetic resistance is an important and major component in the integrated pest management of this disease. To understand molecular mechanisms of root and stem rot resistance in soybeans, the gene and protein expression in hypocotyls and stems of variety Suinong 10 carrying resistance genes Rps1a and Rps2 was investigated by using mRNA differential display reverse transcription PCR and two-dimensional electrophoresis at 0, 0.5, 1, 2, and 4 h after inoculation with P. sojae race 1. The results of the comparison of gene and protein expression showed that at least eight differential fragments at the transcriptional level were related to metabolic pathway, phytoalexin, and signal transduction in defense responses. Sequence analyses indicated that these fragments represented cinnamic acid 4-hydroxylase gene, ATP b gene coding ATP synthase b subunit and ubiquitin-conjugating enzyme gene which upregulated at 0.5 h post inoculation, blue copper protein gene and UDP-N-acetyl-a-D-galactosamine gene which upregulated at 2 h post inoculation, TGA-type basic leucine zipper protein TGA1.1 gene, cyclophilin gene, and 14-3-3 protein gene which upregulated at 4 h post inoculation. Three resistance-related proteins, a-subunit and b-subunit of ATP synthase, and cytochrome P450-like protein, were upregulated at 2 h post inoculation. The results suggested that resistance-related multiple proteins and genes were expressed in the recognition between soybean and P. sojae during zoospore germination, penetration and mycelium growth of P. sojae in soybean.

GmDRR1,a dirigent protein resistant to Phytophthora sojae in Glycine max (L.) Merr.

Soil-borne pathogen Phytophthora sojae is an oomycete that causes devastating damage to soybean yield. To mine original resistant genes in soybean is an effective and environmentally-friend approach controlling the disease. In this study, soybean proteins were extracted from the first trifoliolates infected by predominant P. sojae race 1 and analyzed by twodimensional gel electrophoresis. Nineteen differently-expressed protein spots were detected, and 10 of them were further applied for Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry Assay. One protein containing a dirigent （DIR） domain was identified and belonged to the DIR-b/d family. Therefore, it was named as GmDRR1 （Glycine max Disease Resistance Response 1）. Then, GmDRR1 gene was pathologically confirmed to be involved in the resistant to P. sojae in soybean. GmDRR1-GFP （green fluorescent protein） fusion proteins localized in the cell membrane. qRTPCR results showed GmDRR1 gene expressed differently in P. sojae resistant- and susceptible-soybean cultivars. By the promoter analysis, we found a haplotype H8 was existing in most resistant soybean varieties, while a haplotype H77 was existing in most susceptible soybean varieties. The H77 haplotype had seven SNPs （C to A, G to C, C to A, T to A, T to C, T to C, and T to A） and two single nucleotide insertions. The results supported that the expression difference of GmDRR1 genes between P. sojae resistant- and susceptible-soybean cultivars might depend on the GmDRR1 promoter SNPs. The results suggested that GmDRR1 was a dirigent protein involved in soybean resistant to P. sojae and paved a novel way for investigation of the molecular regulatory mechanism of the defense response to P. sojae in soybean.

Differentially Expressed Genes of Soybean During Infection by Phytophthora sojae

To elucidate the differential gene expression patterns in soybeans during infection by Phytophthora sojae,a cDNA library for suppression subtractive hybridization （SSH） was constructed with cDNAs from soybean cultivar Suinong 10 treated with sterile distilled water as the driver and cDNAs from Suinong 10 inoculated with P.sojae as the tester.A total of 2 067 recombinant colonies from the SSH library were randomly picked,amplified,and sequenced.After discarding 312 poor quality expressed sequence tags （EST）,1 755 high quality ESTs were assembled and edited to 1 384 tentatively unique genes （TUG）,in which,586 showed significant homology to known sequences,and 798 had low homology or no match with the known sequences.A cDNA microarray containing 307 singletons from the 586 TUGs and 222 singletons from the 798 TUGs was developed to characterize differentially expressed cDNAs in the SSH library,and eight cDNAs were identified to be up-regulated after microarray analysis and then confirmed by real-time PCR.They were homologous to the protein 10,and were also related to some proteins in disease resistance response,such as pathogen-related protein,phenylalanine ammonia-lyase,isoflavone reductase,WRKY transcription factor 31,major allergen Pru ar 1,and pleiotropic drug resistance protein 12.Most of the up-regulated cDNAs encode enzymes of phytoalexin biosynthesis and pathogenesis-related proteins involved in plant disease resistance.Here,we fist reported the Pru ar 1 in soybeans.The findings of this research have contributed to better understanding of soybean resistance to P.sojae at the molecular level.

A soybean NAC homolog contributes to resistance to Phytophthora sojae mediated by dirigent proteins

Phytophthora sojae infection severely impairs soybean production. We previously identified a dirigent protein, Gm DRR1(Glycine max Disease Resistant Response 1), that increases soybean resistance to P.sojae. However, the molecular basis of Gm DRR1 function remained largely uncharacterized. In the present study, analysis of Gm DRR1-RNAi, Gm DRR1-overexpressing, and CRISPR/Cas9-derived Gmdrr1 mutant lines revealed that Gm DRR1 expression significantly restricted P. sojae growth. Combining coimmunoprecipitation with liquid chromatography–tandem mass spectrometry revealed a Gm DRR1-interacting protein, Gm DRR2, which is homologous to Gm DRR1. An E-coniferyl alcohol coupling assay indicated that Gm DRR1 promotes the synthesis of(+)-pinoresinol, which helps to protect plants from P. sojae. The Gm NAC1(Glyma.05 G025500) transcription factor bound to the Gm DRR1 promoter both in vitro and in vivo to upregulate Gm DRR1 expression. Soybean resistance to P. sojae was increased by overexpression of Gm NAC1. Our findings suggest a novel signaling pathway involving a NAC transcription factor that mediates soybean resistance to P. sojae. Specifically, Gm NAC1 directly induces Gm DRR1 expression to increase resistance of soybean plants to P. sojae.

Evaluation of Soybean Germplasm from Provinces in Northeast China for Resistance to Phytophthora sojae

Soybean Phytophthora root rot (Phytophthora sojae) is a severe disease all over the world. Soybean germplasm from central and southern China for resistance has been evaluated by American researchers on a large scale. P. sojae has been found frequently in northeast of China in recent years, but not systematic evaluation of soybean germplasm for resistance has occurred there. By means of hypocotyl inoculation, 922 cultivars/lines from northeast of China were screened and evaluated for their response to race 1, and 25 of P. sojae. Generally resistance was less frequent in northeast of China than in central and southern China. Five cultivars/lines were identified that confer resistant responses to race 1, 3, 8, 25 and four additional isolates of P. sojae. These cultivars/lines may provide valuable sources of resistance for future breeding programs.

Fusarium-produced vitamin B6 promotes the evasion of soybean resistance by Phytophthora sojae

Plants can be infected by multiple pathogens concurrently in natural systems. However,pathogen–pathogen interactions have rarely been studied. In addition to the oomycete Phytophthora sojae, fungi such as Fusarium spp. also cause soybean root rot. In a 3-year field investigation, we discovered that P. sojae and Fusarium spp. frequently coexisted in diseased soybean roots. Out of 336 P. sojae–soybean–Fusarium combinations,more than 80% aggravated disease. Different Fusarium species all enhanced P. sojae infection when co-inoculated on soybean. Treatment with Fusarium secreted non-proteinaceous metabolites had an effect equal to the direct pathogen coinoculation. By screening a Fusarium graminearum mutant library, we identified Fusarium promoting factor of Phytophthora sojae infection 1(Fpp1),encoding a zinc alcohol dehydrogenase. Fpp1 is functionally conserved in Fusarium and contributes to metabolite-mediated infection promotion, in which vitamin B6(VB6) produced by Fusarium is key. Transcriptional and functional analyses revealed that Fpp1 regulates two VB6 metabolism genes, and VB6 suppresses expression of soybean disease resistance-related genes. These results reveal that co-infection with Fusarium promotes loss of P. sojae resistance in soybean, information that will inform the sustainable use of diseaseresistant crop varieties and provide new strategies to control soybean root rot.

Life and death:the destiny of Phytophthora sojae determined by a receptor-like kinase

Leucine-rich repeat receptor-like kinases(LRR-RLKs)are well known to act in plant growth,development,and defense responses.Plant LRR-RLKs locate on cell surface to sense and initiate responsive signals to a variety of extracellular stimuli,such as microbe-associated molecular patterns(MAMPs)released from microorganisms.LRR-RLKs are also present in microbes and function in microbial growth and development,but their roles in communicating with hosts are largely unknown.A recent study published in Nature Communications uncovered that a microbial LRR-RLK,PsRLK6,is required for oospore development in the sexual reproduction of Phytophthora sojae,an oomycete pathogen that causes root and stem rot in soybean.Meanwhile,PsRLK6 is recognized as a novel type of MAMP by an unknown plant LRR receptor-like protein and triggers immune responses in soybean,tomato,and Nicotiana benthamiana.The findings reveal dual roles of a pathogen LRR-RLK in determining both life through sexual reproduction and death through triggering plant immunity.

Phytophthora sojae Effector PsAvh240 Inhibits Host Aspartic Protease Secretion to Promote Infection

Plants secrete defense molecules into the extracellular space (the apoplast) to combat attacking microbes. However, the mechanisms by which successful pathogens subvert plant apoplastic immunity remain poorly understood. In this study, we show that PsAvh240, a membrane-localized effector of the soybean pathogen Phytophthora sojae, promotes P. sojae infection in soybean hairy roots. We found that PsAvh240 interacts with the soybean-resistant aspartic protease GmAP1 in planta and suppresses the secretion of GmAP1 into the apoplast. By solving its crystal structure we revealed that PsAvh240 contain six a helices and two WY motifs. The first two a helices of PsAvh240 are responsible for its plasma membrane-localization and are required for PsAvh240's interaction with GmAP1. The second WY motifs of two PsAvh240 molecules form a handshake arrangement resulting in a handshake-like dimer. This dimerization is required for the effector's repression of GmAP1 secretion. Taken together, these data reveal that PsAvh240 localizes at the plasma membrane to interfere with GmAP1 secretion, which represents an effective mechanism by which effector proteins suppress plant apoplastic immunity.

A novel TIR-NBS-LRR gene regulates immune response to Phytophthora root rot in soybean

Phytophthora root rot(PRR),caused by Phytophthora sojae,is a devastating disease of soybean.The NBSLRR gene family is a class of plant genes involved in disease resistance.miRNA mediates plant response to biotic stresses by regulating the expression of target genes at the transcriptional or post-translational level.Glyma.16G135500,encoding an NBS-LRR-type protein,is a target of gma-miR1510 that responds to pathogen infections.We cloned and overexpressed Glyma.16G135500(naming it GmTNL16)and knocked down mi R1510 using short tandem target mimic technology to identify the roles of the GmTNL16/gma-mi R1510 pair in the interaction of soybean and the oomycete.By overexpressing GmTNL16 in transgenic hairy roots of soybean,we showed that biomass of P.sojae was lower in overexpressing hairy roots than in control roots.Thus,miR1510 expression was reduced upon P.sojae infection,reflecting the induced expression of GmTNL16 conferring resistance to P.sojae in soybean.Differentially expressed genes were enriched in plant-pathogen interaction,plant hormone signal transduction,and secondary metabolism by RNA sequencing analyze.In particular,jasmonate and salicylic acid pathway-associated genes,including JAZ,COI1,TGA,and PR,responded to P.sojae infection.All of these results indicate that the GmTNL16/gma-miR1510 pair participates in soybean defense response via the JA and SA pathways.

Resistance Evaluation of Soybean Germplasm from Huanghuai Region to Phytophthora Root Rot

The aim of the study was to establish a set of differential strains and to identify soybean resistant genes to Phytophthora root rot and then to apply those strains for analysis of the resistant genes Rps1a,Rps1c,and Rps1k that soybean cultivars or lines may carry.Virulence formula of 125 Phytophthora sojae isolates were determined using the hypocotyls inoculation technique,the strains,which includ 6 isolates with different virulence formulas,were applied to identify the resistance of 55 soybean cultivars or lines and resistant genes were analyzed using the gene postulating procedure.Eighteen reaction types occurred in 55 cultivars or lines and results of gene postulation indicated that 2 cultivars or lines probably carried gene Rps1c and no cultivar may carry genes Rps1a or Rps1k.A few of soybean cultivars or lines from Huanghuai Region carry Rps genes Rps1a,Rps1c and Rps1k and tend to infect by P.sojae,so resistant cultivars or lines need to be bred and popularized actively.

Impact of Crop Rotation on Pathotype and Genetic Structure of Phythophthora sojae in Fields

To estimate the impact of crop rotation on the pathotype and genetic structure of Phythophthora sojae in fields, 372 isolates of P. sojae were obtained from long-term localisation experimental fields in Heilongjiang Province of China. The hypocotyl inoculation method was used to characterize the virulence of P. sojae on 13 differential cultivars, and the amplified fragment length polymorphism（AFLP） technique was used to analyze difference in the genetic structure of P. sojae. The results indicated that an abundant diversity of genetic structures and pathotypes of P. sojae, a more uniform distribution of pathotypes and less dominance of pathotypes occurred in corn-soybean and wheat-soybean rotation fields than in a continuous soybean mono-cropping field. These findings suggested that P. sojae did not easily become the dominant race in rotation fields, which maintain disease resistance in soybean varieties. Therefore, the results of this study suggested that Phytophthora stem and root rot of soybeans could be effectively controlled by rotating soybeans with non-host crops of corn and wheat.

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 （Ptdlns（3）P） to mediate their translocation into host cells and/or to increase their stability in planta. Since the levels of Ptdlns（3）P in plants are low, we examined whether Phytophthora species may produce Ptdlns（3）P to pro- mote infection. We observed that Ptdlns（3）P-specific GFP biosensors could bind to P. parasitica and P. sojae hyphae dur- ing infection of Nicotiana benthamiana leaves transiently secreting the biosensors, suggesting that the hyphae exposed Ptdlns（3）P on their plasma membrane and/or secreted Ptdlns（3）R Silencing of the phosphatidylinositol 3-kinases （PI3K） genes, treatment with LY294002, or expression of Ptdlns（3）pobinding proteins by P. sojae reduced the virulence of the pathogen on soybean, indicating that pathogen-synthesized Ptdlns（3）P was required for full virulence. Secretion of Ptdlns（3）P-binding proteins or of a PI3P-5-kinase by N. benthamiana leaves significantly increased the level of resist- ance to infection by P. parasitica and P. capsici. Together, our results support the hypothesis that Phytophthora species produce external Ptdlns（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 Avrlb confirm that both the N-terminus and the C-terminus of this effector can bind Ptdlns（3）P.

Transient silencing mediated by in vitro synthesized double-stranded RNA indicates that PsCdc14 is required for sporangial development in a soybean root rot pathogen

In many eukaryotic organisms, Cdcl4 phosphatase regulates multiple biological events during anaphase and is essential for mitosis. It has been shown that Cdcl4 is required for sporulation in the potato blight pathogen Phytophthora infestans; howev- er, the role that the Cdcl4 homolog （PsCdcl4） plays in the soil-borne soybean root rot pathogen P. sojae remains ambiguous. PsCdc14 is highly expressed in spornlation, zoospore, and cyst life stages, but not in vegetative mycelia and infection stages, suggesting that it contributes to asexual reproduction and thus the spread of the disease. Double-stranded RNA （dsRNA） medi- ates gene silencing, a post-transcriptional and highly conserved process in eukaryotes, involving specific gene silencing through degradation of target mRNA. We combined in vitro dsRNA synthesis and a polyethylene glycol-mediated transfor- marion system to construct a dsRNA-mediated transient gene silencing system; and then performed a functional analysis of PsCdcl4 in P. sojae. PsCdc14 mRNA was dramatically reduced in transformants after protoplasts were exposed in in vitro synthesized PsCdc14 dsRNA, resulting in low sporangial production and abnormal development in P. sojae silencing lines. Furthermore, dsRNA-mediated transient gene silencing could enable elucidation of P. sojae rapid gene function, facilitating our understanding of the development and pathogenicity mechanisms of this oomycete fungus.