Evidence that Polymyxa species may infect Arabidopsis thaliana

Polymyxa spp. are obligate biotrophs belonging to the plasmodiophorid group, responsible for transmitting a large number of plant viruses to many crop species. Their obligate nature makes them difficult to study. Controlled environment experiments were used to investigate the potential of infection of Arabidopsis thaliana by Polymyxa spp. to provide a more tractable system. Two ecotypes of Arabidopsis, Columbia and Landsberg erecta, were grown in soils known to be infested with Polymyxa. At the end of a 2-month growth period, both ecotypes were found to harbour Polymyxa-like structures or spores. These findings were confirmed by Polymyxa-specific PCR tests and rDNA sequencing, which positively identified the presence of Polymyxa in the roots of both ecotypes of Arabidopsis. Both Polymyxa graminis and Polymyxa betae were identified. This is the first report of infection of Arabidopsis by Polymyxa spp. and shows the possibility of using this system for studies of infection biology and host-parasite interactions.     

In vitro dual culture of Polymyxa betae in Agrobacterium rhizogenes transformed sugar beet hairy roots in liquid media

Polymyxa betae is a soil-borne protist and an obligate parasite of sugar beet that transmits the beet necrotic yellow vein virus. Sugar beet hairy roots, transformed by Agrobacterium rhizogenes, were inoculated with surface-sterilized root fragments infected by P. betae. After 10 wk in a liquid medium, typical structures of P. betae were observed in this in vitro system. This first in vitro culture of P. betae in liquid medium will contribute to a better understanding of this protist's biology through providing a way to conserve and produce purified isolates of the protist.     

甜菜多粘菌形态发育过程研究

甜菜多粘菌(Polymyxa betae)是一种专性寄生于甜菜根部的低等真菌,可作为甜菜坏死黄脉病毒(BNYVV)的传播介体并与甜菜丛根病的产生有关。对甜菜多粘菌的研究有助于了解其与所传病毒的生物学关系及其在引致甜菜丛根病中所起的作用。本文观察记录了甜菜多粘菌侵染甜菜根系及其在甜菜根细胞中的发育过程,对甜菜多粘菌各主要发育阶段进行了显微镜观察并发现在其游动孢子中有BNYVV粒体存在。根据观察结果并提出了甜菜多粘菌生活史模式图。     

Pseudomonas viridiflava and P. syringae--natural pathogens of Arabidopsis thaliana

We report the isolation and identification of two natural pathogens of Arabidopsis thaliana, Pseudomonas viridiflava and Pseudomonas syringae, in the midwestern United States. P. viridiflava was found in six of seven surveyed Arabidopsis thaliana populations. We confirmed the presence in the isolates of the critical pathogenicity genes hrpS and hrpL. The pathogenicity of these isolates was verified by estimating in planta bacterial growth rates and by testing for disease symptoms and hypersensitive responses to A. thaliana. Infection of 21 A. thaliana ecotypes with six locally collected P. viridiflava isolates and with one P. syringae isolate showed both compatible (disease) and incompatible (resistance) responses. Significant variation in response to infection was evident among Arabidopsis ecotypes, both in terms of symptom development and in planta bacterial growth. The ability to grow and cause disease symptoms on particular ecotypes also varied for some P. viridiflava isolates. We believe that these pathogens will provide a powerful system for exploring coevolution in natural plant-pathogen interactions.     

Identification of a tolerant locus on Arabidopsis thaliana to hypervirulent beet curly top virus CFH strain

The infection of hosts by the geminivirus depends on the interactions between host and viral factors for viral DNA replication, viral gene expression, and the movement of virus throughout the hosts. This work reports that a hypervirulent strain of Beet curly top virus (BCTV) is different in its ability to infect several ecotypes of Arabidopsis thaliana. Symptoms appeared on Arabidopsis ecotypes around 7 to 10 d after inoculation with BCTV-CFH. Symptoms were more severe in ecotype SKKU including severe leaf curling and development of severely deformed and stunted boting compared to Col-O as a lab standard ecotype. One ecotype Cen-O was asymptomatic to BCTV-CFH infection. Studies of viral DNA replication and virus movement in three excised organs of asymptomatic Cen-O demonstrated that BCTV-CFH could replicate viral DNA and move systemically in this ecotype, suggesting that tolerance was due to the blocks of interactions between host and viral factors on symptom development. This asymptomatic phenotype is similar to the mutation of leftward ORFs, especially ORF R2. Genetic analysis of this ecotype Cen-O indicated that tolerance is due to a single recessive locus.     

Studies on the Biology of Polymyxa betae, the Vector of Beet Necrotic Yellow Vein Virus

The development of Polymyxa betae within the roots of a sugar beet variety susceptible to Rhizomania was observed in hydroponic culture over a period of 10 days. Light microscope studies showed that at an average temperature of 20 °C the life cycle of the fungus, containing beet necrotic yellow vein virus (BNYW), was completed within 10 days. A change from the multiplication phase to the survival phase of P. betae became evident. At the beginning of the life cycle the fungus produced mainly zoospores whereas at a later stage plasmodia developed into resting spores. Zoospore density in the nutrient solution reached a maximum between the 5th and 7th day after inoculation and then declined to the initial concentration. The number of zoospores attached to the root surface increased progressively at 48 h intervals, correlated with a parallel increase in BNYW-content of the roots. Light- and fluorescence microscopy revealed that zoospores of P. betae often attach near the point of release and do not move very long distances. In addition it became evident that zoospores may attach to the thallus wall inside the zoosporangium that they have developed in.     

Nucleotide variation at the CHALCONE ISOMERASE locus in Arabidopsis thaliana

An approximately 1.9-kb region encompassing the CHI gene, which encodes chalcone isomerase, was sequenced in 24 worldwide ecotypes of Arabidopsis thaliana (L.) Heynh. and in 1 ecotype of A. lyrata ssp. petraea. There was no evidence for dimorphism at the CHI region. A minimum of three recombination events was inferred in the history of the sampled ecotypes of the highly selfing A. thaliana. The estimated nucleotide diversity theta(TOTAL) = 0.004, theta(SIL) = 0. 005 was on the lower part of the range of the corresponding estimates for other gene regions. The skewness of the frequency spectrum toward an excess of low-frequency polymorphisms, together with the bell-shaped distribution of pairwise nucleotide differences at CHI, suggests that A. thaliana has recently experienced a rapid population growth. Although this pattern could also be explained by a recent selective sweep at the studied region, results from the other studied loci and from an AFLP survey seem to support the expansion hypothesis. Comparison of silent polymorphism and divergence at the CHI region and at the Adh1 and ChiA revealed in some cases a significant deviation of the direct relationship predicted by the neutral theory, which would be compatible with balancing selection acting at the latter regions.     

Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean.

To develop a model system for molecular genetic analysis of plant-pathogen interactions, we studied the interaction between Arabidopsis thaliana and the bacterial pathogen Pseudomonas syringae pv tomato (Pst). Pst strains were found to be virulent or avirulent on specific Arabidopsis ecotypes, and single ecotypes were resistant to some Pst strains and susceptible to others. In many plant-pathogen interactions, disease resistance is controlled by the simultaneous presence of single plant resistance genes and single pathogen avirulence genes. Therefore, we tested whether avirulence genes in Pst controlled induction of resistance in Arabidopsis. Cosmids that determine avirulence were isolated from Pst genomic libraries, and the Pst avirulence locus avrRpt2 was defined. This allowed us to construct pathogens that differed only by the presence or absence of a single putative avirulence gene. We found that Arabidopsis ecotype Col-0 was susceptible to Pst strain DC3000 but resistant to the same strain carrying avrRpt2, suggesting that a single locus in Col-0 determines resistance. As a first step toward genetically mapping the postulated resistance locus, an ecotype susceptible to infection by DC3000 carrying avrRpt2 was identified. The avrRpt2 locus from Pst was also moved into virulent strains of the soybean pathogen P. syringae pv glycinea to test whether this locus could determine avirulence on soybean. The resulting strains induced a resistant response in a cultivar-specific manner, suggesting that similar resistance mechanisms may function in Arabidopsis and soybean.     

Comparative analysis of expressed sequence tags in resistant and susceptible ecotypes of Arabidopsis thaliana infected with cucumber mosaic virus

Arabidopsis thaliana ecotype Columbia (Col-0) is susceptible to the yellow strain of cucumber mosaic virus [CMV(Y)], whereas ecotype C24 is resistant to CMV(Y). Comprehensive analyses of approximately 9,000 expressed sequence tags in ecotypes Col-0 and C24 infected with CMV(Y) suggested that the gene expression patterns in the two ecotypes differed. At 6, 12, 24 and 48 h after CMV(Y) inoculation, the expression of 6, 30, 85 and 788 genes, respectively, had changed in C24, as opposed to 20, 80, 53 and 150 genes in CMV(Y)-infected Col-0. At 12, 24 and 48 h after CMV(Y) inoculation, the abundance of 3, 10 and 55 mRNAs was altered in both ecotypes. However, at 6 h after CMV(Y) inoculation, no genes were co-induced or co-suppressed in both ecotypes. This differential pattern of gene expression between the two ecotypes at an early stage of CMV(Y) infection indicated that the cellular response for resistance may differ from that resulting in susceptibility at the level detectable by the macroarray. According to the expression pattern at various stages of infection, the expression of many genes could be grouped into clusters using cluster analysis. About 100 genes that encode proteins involved in chloroplast function were categorized into clusters 1 and 4, which had a differentially lower expression in CMV(Y)-inoculated C24. The expression of various genes encoding proteins in the endomembrane system belonged to clusters 2 and 4, which were induced in CMV(Y)-inoculated C24 and Col-0 leaves. Characterization of CMV(Y)-altered gene expression in the two ecotypes will contribute to a better understanding of the molecular basis of compatible and incompatible interactions between virus and host plants.     

Mitochondrial DNA variations and nuclear RFLPs reflect different genetic similarities among 23 Arabidopsis thaliana ecotypes

The mitochondrial genome of 23 Arabidopsis thaliana ecotypes was analysed by Southern hybridization in total cellular DNA. Firstly, the extent of divergence between the mitochondrial genomes in closely related lines of one plant species and secondly, the use of mitochondrial versus nuclear RFLPs to determine evolutionary relationships between Arabidopsis ecotype isolates was investigated. Highly divergent stoichiometries of alternative mitochondrial genome arrangements characterize individual ecotypes including the complete loss of a 5 kb region from ecotype Landsberg without apparent effect on plant viability. The genetic similarities between ecotypes suggested by mitochondrial genome arrangements differ from those deduced from 18 nuclear RFLP loci (CAPS markers). Similarity of nuclear RFLP patterns among the 23 Arabidopsis ecotypes neitehr correlates with their geographic origin nor with the observed mitochondrial genome arrangements. A promiscuous mitochondrial sequence insertion previously identified in ecotype Columbia is also found in the nuclear genomes of ecotypes Eifel, Enkheim and Hilversum. Two ecotypes (Eifel and Tabor) displaying identical RFLP patterns at all 18 nuclear loci show differences in both this sequence transfer and a mitochondrial DNA recombination event.     

Changes in Ribonuclease and Glucose-6-Phosphate Dehydrogenase Activities Induced by Beet Necrotic Yellow Vein Virus in Sugar Beet

Activities of host ribonucleases and glucose-6-phosphate dehydrogenase were studied in three cultivars (Monosvalof, Steffi and Rimini) of sugar beet differing in their resistance to beet necrotic yellow vein virus (BNYVV). No differences were found in the susceptibility of cultivars to BNYVV between mechanically inoculated and Polymyxa betae (a natural fungal vector of the virus) infected plants, but the culmination of reproduction curves of BNYVV in mechanically inoculated plants was observed one week earlier than in plants inoculated by means of P. betae. The activities of ribonucleases corresponded with virus multiplication. In roots, activities of ribonucleases reached a maximum at day 7; in leaves, maximum activity was found at day 21 in cv. Monosvalof, and at day 14 in cv. Steffi. The relatively resistant cultivar Rimini showed much lower activities. The activity of glucose-6-phosphate dehydrogenase was only slightly increased at the time of culmination of the BNYVV reproduction curve in cvs. Monosvalof and Steffi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Resistances to turnip mosaic potyvirus in Arabidopsis thaliana.

The responses of a collection of Arabidopsis thaliana ecotypes to mechanical inoculation with turnip mosaic potyvirus were assessed. The virus induced characteristic severe symptoms of infection in systemically infected plants. Resistance was found in four ecotypes: Bay-0, Di-0, Er-0, and Or-0. Enzyme-linked immunosorbent assay results of the resistant ecotypes suggested that ecotypes Di-0, Er-0, and Or-0 actually consist of mixed genotypes with resistances acting at different levels in the virus life cycle. Another form of resistance was found in ecotype Bay-0, for which several lines of evidence indicated an interference with viral cell-to-cell movement in the inoculated leaves.     

Arabidopsis thaliana ecotype Cvi shows an increased tolerance to photo-oxidative stress and contains a new chloroplastic copper/zinc superoxide dismutase isoenzyme.

A new chloroplastic Cu/Zn-superoxide dismutase (SOD) isoenzyme was identified in Arabidopsis thaliana ecotype Cvi. Genetic analyses indicated that the new isoenzyme was encoded by a Cvi-specific allele of Csd2 that was named Csd2-2. Paraquat treatments of A. thaliana ecotypes Ler and Cvi resulted in higher levels of chloroplastic Cu/Zn-SOD activity in Cvi, suggesting that the Cvi isoenzyme has a higher stability and/or turnover rate than the Ler variant under photo-oxidative conditions. In addition, Cvi showed a higher tolerance to paraquat treatments. Hybrid plant populations expressing Csd2-2 also exhibited an increased tolerance, suggesting that the Cvi isoenzyme is one of the factors that contribute to a better fitness in photo-oxidative stress conditions.     

Susceptibility and symptom development in Arabidopsis thaliana to Tobacco mosaic virus is influenced by virus cell-to-cell movement

To identify host factors that regulate susceptibility to Tobacco mosaic virus (TMV), 14 Arabidopsis thaliana ecotypes were screened for their ability to support TMV systemic movement. The susceptibility phenotypes observed included one ecotype that permitted rapid TMV movement accompanied by symptoms, nine ecotypes that allowed a slower intermediate rate of systemic movement without symptoms, and four ecotypes that allowed little or no systemic TMV movement. Molecular comparisons between ecotypes representing the rapid (Shahdara), intermediate (Col-1), and slow (Tsu-1) movement phenotypes revealed a positive correlation between the ability of TMV to move cell to cell and its speed of systemic movement. Additionally, protoplasts prepared from all three ecotypes supported similar levels of TMV replication, indicating that viral replication did not account for differences in systemic movement. Furthermore, induction of the pathogenesis-related genes PR-1 and PR-5 occurred only in the highly susceptible ecotype Shahdara, demonstrating that reduced local and systemic movement in Col-1 and Tsu-1 was not due to the activation of known host defense responses. Genetic analysis of F2 progeny derived from crosses made between Shahdara and Tsu-1 or Col-1 and Tsu-1 showed the faster cell-to-cell movement phenotypes of Shahdara and Col-1 segregated as single dominant genes. In addition, the Shahdara symptom phenotype segregated independently as a single recessive gene. Taken together, these findings suggest that, within Arabidopsis ecotypes, at least two genes modulate susceptibility to TMV.     

Variations in xanthophyll composition in etiolated seedlings of Arabidopsis thaliana correlate with protochlorophyllide accumulation

Protochlorophyllide (Pchlide) accumulation and xantophyll composition were studied in 5-day old etiolated seedlings of three ecotypes of Arabidopsis thaliana: Columbia (Col-0), Landsberg erecta (Ler) and Wassiliewska (Ws). The total Pchlide level as measured by fluorescence spectroscopy varied significantly between ecotypes. A rapid HPLC method revealed quantitative differences in carotenoid composition. It was found that in the Ler ecotype any enhanced accumulation of Pchlide correlates with an increased level of lutein, suggesting the role of enzymes involved in lutein synthesis in cross-regulation between chlorophyll and carotenoid biosynthetic pathways. The function of the dark-accumulated carotenoid pool in seedling de-etiolation is discussed.