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Nonhost Interaction of Phytophthora sojae and Arabidopsis thaliana and Genetic Analysis of a Susceptible Mutant

Qiuping Liu,Hua Cao,Maojin Yao,Ying Ma,Binsheng Deng,Junli Quan,Weixing Shan,

植物学报 , 2010,
Abstract: Phytophthora sojae and Arabidopsis thaliana were used in this study as a nonhost plant-oomycete interaction system to investigate the genetic basis of nonhost resistance against oomycete pathogens in plants. A collection of more than 40 000 T3 A. thaliana T-DNA mutant plants representing 12 000 independent insertion lines were screened by inoculating detached leaves with P. sojae zoospores, and the susceptible mutant was re-confirmed by pathogen inoculation and cytological characterization. A number of P. sojae-susceptible A. thaliana mutants were successfully obtained, and one of them, mutant 581-51, was shown to be stably susceptible to P. sojae infection. Water-soaked lesions formed on the detached leaves within 3 days, as did oospores and sporangia 4–5 days after inoculation with P. sojae zoospores. Cytological characterization revealed the formation of haustoria-like structures. Southern analysis showed the presence of four T-DNA insertion events in the mutant. Genetic analysis indicated that the susceptibility to infection by the nonhost pathogen P. sojae in the mutant 581-51 was likely controlled by a single recessive gene.
Green fluorescent protein (GFP) as a vital marker for studying the interaction of Phytophthora sojae and soybean
XiaoRen Chen,BaoPing Cheng,XinLe Wang,SuoMeng Dong,YongLin Wang,XiaoBo Zheng,YuanChao Wang
Chinese Science Bulletin , 2009, DOI: 10.1007/s11434-009-0417-7
Abstract: Transgenic Phytophthora sojae strains that produce green fluorescent protein (GFP) were obtained after stable DNA integration using the Hsp70 promoter and the Ham34 terminator of Bremia lactucae. The expression of GFP during different developmental stages of P. sojae was observed using fluorescent microscopy. Based on this reporter system, the histopathologic events caused by the pathogen in soybean leaves, hypocotyls and roots were monitored. Meanwhile, the difference in resistance between different soybean cultivars against P. sojae was analyzed microscopically in roots. The results indicate that GFP can be stably expressed in zoosporangia, zoospores, cysts, hyphae and oospores of P. sojae. Using the GFP marker, the infecting pathogens in leaves, hypocotyls and roots of host could be distinctly visualized. The germ tube length of cysts germinating on the roots of resistant cultivar Nannong 8848 was longer than that on the roots of susceptible cultivar Hefeng 35. These results show for the first time that this eukaryotic reporter can be used in P. sojae as a stable and vital marker, allowing the study of genetics of this hemibiotrophic pathogen.
EMS mutagenesis analysis of Phytophthora sojae

HA Xia,HU Zhong-Hui,WANG Lei,QUAN Jun-Li,SHAN Wei-Xing,

菌物学报 , 2010,
Abstract: The commonly used chemical mutagen EMS (ethylmethane sulfonate) was employed to create a mutant collection by treating the encysted zoospores of Phytophthora sojae and the spore germination was used to optimize the mutagenesis condition. A total of 640 single-oospore mutant lines were acquired, among which 50% of them showed a wide range of morphological changes. As to the production of oospores, 8.13% mutants produced more, 20.41% less, and 27.82% very few or no oospores as compared with the wild type, respectively, and 43.64% were similar to the wild type. The quality of the mutant collection was further confirmed by successful identification of 9 mutations in PsPMA1 (Plasma Membrane H+-ATPase 1) gene from 320 P. sojae mutants. Thus, the estimated maximum frequency of EMS induced mutation is 1 per 115kb of sequence. The created P. sojae mutant collection will be useful for functional genomic analysis of P. sojae.
A Myb Transcription Factor of Phytophthora sojae, Regulated by MAP Kinase PsSAK1, Is Required for Zoospore Development  [PDF]
Meng Zhang, Jing Lu, Kai Tao, Wenwu Ye, Aining Li, Xiaoyun Liu, Liang Kong, Suomeng Dong, Xiaobo Zheng, Yuanchao Wang
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0040246
Abstract: PsSAK1, a mitogen-activated protein (MAP) kinase from Phytophthora sojae, plays an important role in host infection and zoospore viability. However, the downstream mechanism of PsSAK1 remains unclear. In this study, the 3'-tag digital gene expression (DGE) profiling method was applied to sequence the global transcriptional sequence of PsSAK1-silenced mutants during the cysts stage and 1.5 h after inoculation onto susceptible soybean leaf tissues. Compared with the gene expression levels of the recipient P. sojae strain, several candidates of Myb family were differentially expressed (up or down) in response to the loss of PsSAK1, including of a R2R3-type Myb transcription factor, PsMYB1. qRT-PCR indicated that the transcriptional level of PsMYB1 decreased due to PsSAK1 silencing. The transcriptional level of PsMYB1 increased during sporulating hyphae, in germinated cysts, and early infection. Silencing of PsMYB1 results in three phenotypes: a) no cleavage of the cytoplasm into uninucleate zoospores or release of normal zoospores, b) direct germination of sporangia, and c) afunction in zoospore-mediated plant infection. Our data indicate that the PsMYB1 transcription factor functions downstream of MAP kinase PsSAK1 and is required for zoospore development of P. sojae.
Molecular Detection of Phytophthora sojae

LIU Chun-Lai,YANG Ming-Xiu,WEN Jing-Zhi,

微生物学通报 , 2007,
Abstract: An oligonucleotide primer pair was designed and synthesized after comparison and homological analysis of rDNA ITS sequences among Phytophthora sojae,its related Phytophthora species,and allied fungal and bacterial species from GenBank.PCR amplifications were carried out for 140 isolates including Phytophthora sojae.It showed that only isolates of Phytophthora sojae can be amplified and a special fragment of 288bp were produced by the primers.These primers were used to detect Phytophthora sojae in pure culture,inoculated diseased soybean plants,and inoculated soil samples.The detection protocol has good sensitivity to diseased tissues.
Races of Phytophthora sojae in Iran  [PDF]
Abbas Mohammadi,Azizollah Alizadeh,Mansore Mirabolfathey,Nasrin Nooras Mofrad
Pakistan Journal of Biological Sciences , 2008,
Abstract: Phytophthora root and stem rot of soybean is a destructive disease of soybean in Iran. Races 1 and 3 of pathogen have already been reported from two major growing regions of the crop, Lorestan and Golestan provinces. In a survey during 2004-2005, 142 isolates of P. sojae were recovered from infected plants and naturally infested soil samples using selective media and soybean leaf baiting technique. The majority of tested isolates (110 isolates) belonged to race one of P. sojae and 32 isolates belonged to race 3. ITS region of 23 isolates were amplified with specific primers Ps1 and Ps2. Sequences of this regions were similar to other gene banks sequences except two isolates from China. This survey showed low diversity in Iranian population of P. sojae.
Identification and expression analysis of apoptosis-related genes in soybean stem and root rot pathogen Phytophthora sojae

陈琳琳,耿雪晶,王雯,张雄,窦道龙,李洪连,Chen Linlin,Geng Xuejing,Wang Wen,Zhang Xiong,Dou Daolong,Li Honglian
- , 2018, DOI: 10.13802/j.cnki.zwbhxb.2018.2017017
Abstract: 为探讨大豆疫霉Phytophthora sojae细胞凋亡潜在的调控机制,根据已知的细胞凋亡蛋白,利用在线工具BLASTP、pFAM和SMART在大豆疫霉蛋白组数据库中鉴定细胞凋亡同源蛋白并构建其进化树,通过转录组数据和实时荧光定量PCR技术分析细胞凋亡相关基因在大豆疫霉生长、发育及侵染不同时期的表达情况。结果显示:在大豆疫霉中共鉴定到13个细胞凋亡同源蛋白,包括核酸内切酶G (PsNUC1)、细胞色素c (PsCYCS)、凋亡诱导因子(PsAIF)、丝氨酸蛋白酶(PsHtrA-1、PsHtrA-2和PsHtrA-3)、多聚ADP核糖聚合酶(PsPARP-1、PsPARP-2和PsPARP-3)和TatD核酸酶(PsTatD1、PsTatD2、PsTatD3和PsTatD4)。在进化上,PsNUC1、PsCYCS、PsAIF、PsHtrA-1、PsPARP-1、PsPARP-2、PsPARP-3、PsTatD1和PsTatD2与人及秀丽隐杆线虫Caenorhabditis elegans的同源蛋白亲缘关系较近,而与真菌相关蛋白亲缘关系较远,PsHtrA-2、PsHtrA-3、PsTatD3和PsTatD4与酿酒酵母Saccharomyces cerevisiae相关蛋白相似度更高,说明大豆疫霉细胞凋亡蛋白在进化中发生了较大变异。大豆疫霉细胞凋亡相关基因PsHtrA-1和PsRARP-1在孢子囊阶段诱导表达,PsHtrA-2和PsRARP-2在游动孢子阶段上调表达,PsAIF、PsHtrA-3、PsRARP-1和PsRARP-2在侵染阶段明显诱导表达,PsCYCS在侵染阶段下调表达。细胞凋亡相关基因在大豆疫霉不同阶段的表达模式有较大差异,说明细胞凋亡在大豆疫霉生长、发育及致病过程中具有重要作用。
To investigate the possible mechanisms of apoptosis in soybean stem and root rot pathogen Phytophthora sojae, the apoptosis-related proteins in P. sojae were identified based on known apoptosisrelated protein sequences by using BLASTP, pFAM and SMART. Apoptosis-related homologous neighbor-joining phylogenetic trees were constructed using Mega 4.0, and apoptosis-related genes transcription levels in P. sojae growth, development and infection were examined by qRT-PCR and transcriptome data. The results showed that endonuclease G (PsNUC1), cytochrome c (PsCYCS), apoptosis inducing factor (PsAIF), serine protease (PsHtrA-1, PsHtrA-2 and PsHtrA-3), poly (ADP-ribose) polymerase (PsPARP-1, PsPARP-2 and PsPARP-3) and TatD nuclease (PsTatD1, PsTatD2, PsTatD3 and PsTatD4) were identified by bioinformatics methods in P. sojae. In the phylogenetic trees, PsNUC1, PsCYCS, PsAIF, PsHtrA-1, PsPARP-1, PsPARP-2, PsPARP-3, PsTatD1 and PsTatD2 in P. sojae were close to homolog proteins from Homo sapiens and Caenorhabditis elegans, rather than fungi, while PsHtrA-2, PsHtrA-3, PsTatD3 and PsTatD4 were closer to related proteins from Saccharomyces cerevisiae. These results suggested that apoptosis genes had undergone great changes in P. sojae evolution. Transcriptome data and real-time PCR analysis revealed that PsHtrA-1 and PsRARP-1 were highly expressed in P. sojae sporangia, and both PsHtrA-2 and PsRARP-2 were highly upregulated in zoospores. Four apoptosis-related genes, including PsAIF, PsHtrA-3, PsRARP-1 and PsRARP-2, showed a relatively higher expression level during infection stages, while PsCYCS was downregulated during infection stages. The apoptosis might function in P. sojae growth and pathogenicity.
Autonomous movement, root location and infection by sporangial and cyst derived zoospores of Phytophthora cryptogea isolates in water  [cached]
Indian Phytopathology , 2011,
Abstract: The ability of cyst derived zoospores (CDZs) and sporangial derived zoospores (SDZs) to move autonomously in water in model mazes and linear troughs and to infect roots of vinca ( Catharanthus roseus) seedlings at fixed distances from a point of introduction were assessed at 4 h for two isolates of Phytophthora cryptogea (FWDM4 and FDM51). The study showed that both CDZs and SDZs infected roots at distances up to 215 mm (in model mazes) and 320 mm (in linear troughs), and were also able to move the same distances in controls without seedlings. A concentration of 104 zoospores/ml when used at the point of introduction in linear troughs, SDZs and CDZs of the isolate, FDM51 moved actively and infected roots to a distance of 320 mm. SDZs and CDZs of FWDM4 infected seedlings at distances of 240 mm and 320 mm respectively and both the isolates were recovered at 320 mm without seedlings. The results indicate that the CDZs of Phytophthora cryptogea can serve as effective dispersal and infection units in water and suggests that zoospores may be able to move autonomously to greater distances in a non-restraining medium than previously thought.
Secondary zoospores in the algal endoparasite Maullinia ectocarpii (Plasmodiophoromycota)
Parodi,Elisa R.; Cáceres,Eduardo J.; Westermeier,Renato; Müller,Dieter G.;
Biocell , 2010,
Abstract: the present paper deals with the ultrastructure of zoospores produced by the plasmodiophorid maullinia ectocarpii, living in the marine algal host ectocarpus siliculosus. the zoospores described here are very similar to secondary zoospores of polymyxa graminis and phagomyxa sp.(the latter an algal endoparasite, also). our results indicate that m. ectocarpii produces two types of plasmodia, and suggest that is a species with a complete life cycle, as it is known for all the plasmodiophormycota that have been studied. sporogenic and sporangial plasmodia produce, respectively, primary zoospores with parallel flagella within thick walled resting sporangia, and secondary zoospores with opposite flagella within thin walled sporangia.
Secondary zoospores in the algal endoparasite Maullinia ectocarpii (Plasmodiophoromycota)
Elisa R. Parodi,Eduardo J. Cáceres,Renato Westermeier,Dieter G. Müller
Biocell , 2010,
Abstract: The present paper deals with the ultrastructure of zoospores produced by the plasmodiophorid Maullinia ectocarpii, living in the marine algal host Ectocarpus siliculosus. The zoospores described here are very similar to secondary zoospores of Polymyxa graminis and Phagomyxa sp.(the latter an algal endoparasite, also). Our results indicate that M. ectocarpii produces two types of plasmodia, and suggest that is a species with a complete life cycle, as it is known for all the Plasmodiophormycota that have been studied. Sporogenic and sporangial plasmodia produce, respectively, primary zoospores with parallel flagella within thick walled resting sporangia, and secondary zoospores with opposite flagella within thin walled sporangia.
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