Overlapping expression patterns and differential transcript levels of phosphate transporter genes in arbuscular mycorrhizal,P<Subscript>i</Subscript>-fertilised and phytohormone-treated <Emphasis Type="Italic">Medicago truncatula</Emphasis> roots

A microarray carrying 5,648 probes of Medicago truncatula root-expressed genes was screened in order to identify those that are specifically regulated by the arbuscular mycorrhizal (AM) fungus Gigaspora rosea, by P_i fertilisation or by the phytohormones abscisic acid and jasmonic acid. Amongst the identified genes, 21% showed a common induction and 31% a common repression between roots fertilised with P_i or inoculated with the AM fungus G. rosea, while there was no obvious overlap in the expression patterns between mycorrhizal and phytohormone-treated roots. Expression patterns were further studied by comparing the results with published data obtained from roots colonised by the AM fungi Glomus mosseae and Glomus intraradices, but only very few genes were identified as being commonly regulated by all three AM fungi. Analysis of P_i concentrations in plants colonised by either of the three AM fungi revealed that this could be due to the higher P_i levels in plants inoculated by G. rosea compared with the other two fungi, explaining that numerous genes are commonly regulated by the interaction with G. rosea and by phosphate. Differential gene expression in roots inoculated with the three AM fungi was further studied by expression analyses of six genes from the phosphate transporter gene family in M. truncatula. While MtPT4 was induced by all three fungi, the other five genes showed different degrees of repression mirroring the functional differences in phosphate nutrition by G. rosea, G. mosseae and G. intraradices. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Functional characterization of the pollen-specific <Emphasis Type="Italic">SBgLR</Emphasis> promoter from potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

SBgLR (Solanum tuberosum genomic lysine-rich) gene was isolated from a potato genomic library using SB401 (S. berthaultii 401) cDNA as probe. RT-PCR analysis of SBgLR gene expression profile and microscopic analysis of green fluorescent protein (GFP) expression in tobacco plants transformed with SBgLR promoter-GFP reporters indicate that SBgLR is a pollen-specific gene. A series of 5′deletions of SBgLR promoter were fused to the β-glucuronidase (GUS) gene and stably introduced into tobacco plants. Histochemical and quantitative assays of GUS expression in transgenic plants allowed us to localize an enhancer of SBgLR promoter to the region −345 to −269 relative to the translation start site. This 76 bp (−345 to −269) fragment enhanced GUS expression in leaves, stems and roots when fused to −90/+6 CaMV 35S minimal promoter. Deletion analysis showed that a cis-element, which can repress gene expression in root hairs, was located in the region −345 to −311. Further study indicated that the −269 to −9 region was sufficient to confer pollen-specific expression of GFP when fused to CaMV 35S enhancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Authors Zhihong Lang and Peng Zhou contributed equally to this work.     

Exopolysaccharide from surface-liquid culture of <Emphasis Type="Italic">Clonostachys</Emphasis><Emphasis Type="Italic">rosea</Emphasis> originates from autolysis of the biomass

We describe the purification and chemical characterization of galactomannans that appear both in the biomass and the culture broth during surface-liquid culture of the fungus Clonostachys rosea, a common facultative saprophyte that has potential to be used as a biological control agent against several plant pathogenic fungi, insects and nematodes. The galactomannans from both sources had comparable ratios of Man, Gal and Glc and the similarity were confirmed by ¹H, ¹³C NMR, HMQC, and COSY spectra. We propose that the galactomannan in the culture broth originates from autolysis of the biomass, based not only on the similarity that it has with the galactomannan extracted from the biomass but also on the fact that its concentration increased rapidly after glucose depletion from the medium, when biomass concentration was falling. Polysaccharides from C. rosea have not previously been characterized; we show that the characteristics of the galactomannans are consistent with those that have been reported for other members of the Bionectriaceae, the family to which C. rosea belongs.     

Development of a GFP-Expressing Aspergillus flavus Strain to Study Fungal Invasion, Colonization, and Resistance in Cottonseed

Cotton bolls were inoculated with a green fluorescent protein (GFP)-expressing Aspergillus flavus (strain 70) to monitor fungal growth, mode of entry, colonization of cottonseeds, and production of aflatoxins. The GFP strain and the wild-type did not differ significantly in pathogen aggressiveness as indicated by similar reductions in inoculated locule weight. GFP fluorescence was at least 10 times higher than the blue green yellow fluorescence (BGYF) produced in response to infection by A. flavus. The GFP produced by the strain made it possible to identify and monitor specific plant tissues colonized by the fungus. For example, the inner seed coat and cotyledon were colonized by the fungus within 72 h of inoculation and the mode of entry was invariably through the porous chalazal cap in intact seeds. The amount of GFP fluorescence was shown to be an indicator of fungal growth, colonization and, to some extent, aflatoxin production. The A. flavus strain expressing GFP should be very useful for rapidly identifying cotton lines with enhanced resistance to A. flavus colonization developed through genetic engineering or traditional plant breeding. In addition, development of GFP expressing A. flavus strain provides an easy and rapid assay procedure for studying the ecology, etiology, and epidemiology of cotton boll rot caused by A. flavus resulting in aflatoxin contamination. The U.S. Government’s right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Ralstonia solanacearum colonization of tomato roots infected by Meloidogyne incognita

Bacterial wilt, caused by Ralstonia solanacearum, is one of the most serious diseases of tomato (Solanum lycopersicum). Concomitant infection of R. solanacearum and root‐knot nematode Meloidogyne incognita increases the severity of bacterial wilt in tomato, but the role of this nematode in disease complexes involving bacterial pathogens is not completely elucidated. Although root wounding by root‐knot nematode infection seems to play an important role, it might not entirely explain the increased susceptibility of plants to R. solanacearum. In the present study, green fluorescent protein (GFP)‐labelled R. solanacearum distribution was observed in the root systems of the tomato cultivar Momotaro preinoculated with root‐knot nematode or mock‐inoculated with tap water. Fluorescence microscopy revealed that GFP‐labelled R. solanacearum mainly colonized root‐knot nematode galls, and little or no green fluorescence was observed in nematode‐uninfected roots. These results suggest that the gall induced by the nematode is a suitable location for the growth of R. solanacearum. Thus, it is crucial to control both R. solanacearum and root‐knot nematode in tomato production fields to reduce bacterial wilt disease incidence and effects.     

Purification and cloning of a novel serine protease from the nematode-trapping fungus <Emphasis Type="Italic">Dactylellina varietas</Emphasis> and its potential roles in infection against nematodes

From the culture filtrate of the fungus Dactylellina varietas (syn. Dactylella varietas), an extracellular protease (designed Dv1) was purified by cation exchange and hydrophobic interaction chromatography. The purified protease showed a molecular mass of approximately 30 kDa and displayed an optimal activity at pH 8 and 60.5°C (more than 20 min). This protease could degrade a broad range of substrates including casein, gelatin, BSA (bovine serum albumin), and nematode cuticle. However, its proteolytic activity was highly sensitive to the serine protease inhibitor Phenylmethylphonylfuoride (1 mM), indicating that it belongs to the serine-type peptidase group. This protease could immobilize the free-living nematodes Panagrellus redivivus and Caenorhabditis elegans and hydrolyze the purified cuticle of P. redivivus, suggesting it may play a role in infection against nematodes. The encoding gene of Dv1 and its promoter sequence were cloned using degenerate primers and the DNA walking technology. Its open-reading frame contains 1,224 base pairs and without any intron. The deduced amino-acid sequence shared low identity to serine proteases from other nematode-trapping fungi. Our report identified a novel pathogenic protease from the nematode-trapping fungus D. varietas, and the three-dimensional structure of this protease was predicted using the Swiss-Prot method. Jinkui Yang and Lianming Liang contributed equally to this work.     

Cloning and characterization of an extracellular serine protease from the nematode-trapping fungus <Emphasis Type="Italic">Arthrobotrys conoides</Emphasis>

An extracellular serine protease (Ac1) with a molecular mass of 35 kDa was purified from the nematode-trapping fungus Arthrobotrys conoides. The optimum activity of Ac1 is at pH 7.0 and 53.2°C (over 20 min). Ac1 can degrade a broad range of substrates including casein, gelatin, bovine serum albumin, collagen, and nematode cuticles. Moreover, the enzyme can immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, indicating Ac1 may be involved in infection against nematodes. The encoding gene of Ac1 contains one intron of 60-bp and two exons encoding a polypeptide of 411 amino acid residues. The deduced polypeptide sequence of Ac1 showed a high degree of similarity to two previously reported serine proteases PII and Mlx from other nematode-trapping fungi (81% aa sequence identity). However, three proteases Ac1, Aoz1 and Mlx showed optimum temperatures at 53.2, 45 and 65°C, respectively. Compared to PII, Ac1 appears to have a significantly higher activity against gelatin, bovine serum albumin, and non-denatured collagen. Moreover, our bioassay experiments showed that Ac1 is more effective at immobilizing P. redivivus than B. xylophilus.     

Infection of plant-parasitic nematodes by Paecilomyces lilacinus and Monacrosporium lysipagum

Studying the mode of infection of a biocontrol agent is important in order to assess its efficiency. The mode and severity of infection of nematodes by a soil saprophyte Paecilomyces lilacinus (Thom) Samson and a knob-producing nematode trapping fungus Monacrosporium lysipagum (Drechsler) Subram were studied under laboratory conditions using microscopy. Infection of stationary stages of nematodes by P. lilacinus was studied with three plant-parasitic nematodes Meloidogyne javanica (Treub) Chitwood, Heterodera avenae Wollenweber and Radopholus similis (Cobb) Thorne. Paecilomyces lilacinus infected eggs, juveniles and females of M. javanica by direct hyphal penetration. The early developed eggs were more susceptible than the eggs containing fully developed juveniles. As observed by transmission electron microscopy, fungal hypha penetrated the M. javanica female cuticle directly. Paecilomyces lilacinus also infected immature cysts of H. avenae including eggs in the cysts and the eggs of R. similis. Trapping and subsequent killing of mobile stages of nematodes by M. lysipagum were studied with the above three nematodes. In addition, plant-parasitic nematodes Pratylenchus neglectus (Rensch) Chitwood and Oteifa and Ditylenchus dipsaci (Kuhn) Filipjev were tested with M. lysipagum. This fungus was shown to infect mobile stages of all the plant-parasitic nematodes. In general, juveniles except those of P. neglectus, were more susceptible to the attack than adults.     

Stages of Infection during the Tripartite Interaction between Xenorhabdus nematophila, Its Nematode Vector, and Insect Hosts

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.     

Role of an extracellular neutral protease in infection against nematodes by <Emphasis Type="Italic">Brevibacillus laterosporus</Emphasis> strain G4

Proteases have been proposed as virulence factors in microbial pathogenicity against nematodes. However, what kinds of extracellular proteases from these pathogens and how they contribute to the pathogenesis of infections against nematode in vivo remain largely unknown. A previous analysis using a strain with a deletion in an extracellular alkaline protease BLG4 gene from Brevibacillus laterosporus demonstrated that BLG4 was responsible for the majority of nematicidal activity by destroying host’s cuticle. In recent studies, a neutral protease NPE-4, purified from the mutant BLG4–6, was found to be responsible for the majority of the remaining EDTA-inhibited protease activity. However, the purified NPE-4 and recombinant NPE-4 in a related species Bacillus subtilis showed little nematicidal activity in vitro and were unable to degrade the intact cuticle of the host. It is interesting to note that the addition of NPE-4 improved the pathogenicity of crude enzyme extract from wild-type B. laterosporus but had no effect on the BLG4-deficient mutant. This result suggests that NPE-4 functions in the presence of protease BLG4. Moreover, NPE-4 could degrade proteins from the inner layer of purified cuticles from nematode Panagrellus redivivus in vitro. These results indicated that the two different bacterial extracellular proteases might play differential roles at different stages of infection or a synthetic role in penetration of nematode cuticle in B. laterosporus. This is among the first reports to systematically evaluate and define the roles of different bacterial extracellular proteases in infection against nematodes.     

Interactions between Escherichia coli and the plant‐parasitic nematode Meloidogyne javanica

Aims: To determine the potential of the plant‐parasitic nematode Meloidogyne javanica to serve as a temporary reservoir for Escherichia coli. Methods and Results: The adhesion to and persistence of E. coli on the surface of M. javanica were evaluated at different times and temperatures. A pure culture of green fluorescent protein (GFP) tagged E. coli was mixed with ca. 1000 J2 M. javanica for 2 h at 25°C. The nematodes were then washed and the rate of the adhesion of the bacteria to the nematodes was determined by counting the viable nematode‐associated E. coli, and by fluorescence microscopy. A dose‐dependent adhesion rate was observed only at a bacterium to nematode ratio of 10⁴–10⁶ : 1. The adhesion of E. coli to the nematodes was also tested over a 24 h‐period at 4°C, 25°C and 37°C. At 4°C and 37°C, maximal adhesion was observed at 5 h; whereas at 25°C, maximal adherence was observed at 8 h. Survival experiments showed that the bacteria could be detected on the nematodes for up to 2 weeks when incubated at 4°C and 25°C, but not at 37°C. Conclusions: Under laboratory conditions, at 4°C and 25°C, M. javanica could serve as a temporary vector for E. coli for up to 2 weeks. Significance and Impact of the Study: These findings support the hypothesis that, in the presence of high concentrations of E. coli, M. javanica might serve as a potential vehicle for the transmission of food‐borne pathogens.     

Attraction of Pinewood Nematode to Endoparasitic Nematophagous Fungus Esteya vermicola

The investigations on attraction of nematodes to nematophagous fungi have mostly dealt with the nematode-trapping species. Esteya vermicola is the endoparasitic fungus of pinewood nematode (PWN) with high infection activity. In the present study, the attraction of PWNs to E. vermicola was investigated. It was confirmed that the living mycelia and exudative substances of E. vermicola were attractive to PWN. Compared with the nematode-trapping fungus A. brochopaga as well as nematode-feeding fungus B. cinerea, E. vermicola showed the significantly strongest attraction ability to nematode. It therefore appeared that the attraction ability reflects the dependence of the fungi on nematodes for nutrients. Furthermore, a new method was developed and used in the study to confirm the effect of volatile substances for the attraction of nematode to fungi. The results suggested that the attractive substances were consisted of avolatile exudative and volatile diffusing compounds.     

灰葡萄孢菌过氧化物酶体及细胞核的荧光蛋白标记

[目的]对灰葡萄孢菌(Botrytis cinerea)的细胞核和过氧化物酶体进行荧光蛋白标记,为研究其生长发育和侵染过程中细胞结构和细胞器动态提供基础.[方法]以绿色荧光蛋白(GFP)和红色荧光蛋白(DsRED、mCherry)为报告基因,利用根癌农杆菌介导转化(Agrobacterium tumefaciens m...     

Direct visualization of bacterial infection process in nematode hosts by an improved immunocytochemical method

Our previous work on Brevibacillus laterosporus G4 had demonstrated that an extracellular protease BLG4 played a key role as a pathogenic factor in bacterial infection of nematodes. In this study, we developed an improved method for the immunofluorescent localization of protease BLG4 to directly visualize the bacterial infection process in a nematode host and demonstrate the involvement of the protease in penetrating nematode cuticles in vivo. This work supplies a new basic knowledge that elucidates the microbial infection processes in nematodes. B.Y. Tian and C.R. Ke have contributed equally to this work.     

Cloning, expression and deletion of the cuticle-degrading protease BLG4 from nematophagous bacterium Brevibacillus laterosporus G4

Brevibacillus laterosporus G4, which was isolated from soil sample, kills free-living nematodes (Panagrellus redivius) and plant-parasite nematodes (Bursaphelenchus xylophilus) and degrades their cuticle in previous bioassay. Our works for B. laterosporus G4 had demonstrated that an extracellular alkaline protease BLG4 played a key role as a pathogenic factor in infection against nematode. In this study, the nematicidal activity of BLG4 was further verified by an in vitro assay with purified recombinant BLG4. The encoding gene of BLG4 was cloned and showed high degree of homology with the subtilisin subclass of serine protease gene and another reported cuticle-degrading protease gene from nematophagous bacterium Bacillus sp. B16. Deletion of BLG4 by homologous recombinant had a significant effect on the pathogenicity of B. laterosporus. In infection assays the BLG4-deficient strain (BLG4-6) lost about 50% of its nematocidal activity and in toxicity tests the mortality rate of nematodes decreased with ∼56% in comparison to wild-type strain. This is the first report analyzing the function of a subtilisin enzyme involved in bacterium against nematode at the molecular level, and it is possible to use B. laterosporus as a model to study host-parasite interaction and to gain detailed knowledge of the infection process.     

Inhabitancy and Colonization on Healthy Rice Plants by Glomerella cingulata

An omnivorous phytopathogenic fungus, Glomerella cingulata, was isolated from rice plants in Japan. We evaluated the effect of the fungus as an inoculum source for anthracnose to other plants. Leaf sheaths of rice plants were monitored in mid‐June and mid‐July for 2 years to evaluate the quantitative inhabitancy of G. cingulata in the fungal community. The pathogenicity of G. cingulata to 20 plant leaves was elucidated. A hygromycin B‐resistant green fluorescent protein (GFP) mutant of the fungus was used to observe fungal infection and development processes on rice plants. Glomerella cingulata was detected on rice sheaths in mid‐June at a low frequency, but was not detected in mid‐July. The fungus has a broad pathogenic spectrum, whereas development of the GFP mutant in tissues of rice plants was limited. Thus, the effect of G. cingulata inhabiting rice plants as a source of inoculum for other crops is likely to be low, although the fungal potential to infect various plants was ascertained.     

Altered expression of the<Emphasis Type="Italic"> Arabidopsis</Emphasis> ortholog of<Emphasis Type="Italic"> DCL</Emphasis> affects normal plant development

The DCL (defective chloroplasts and leaves) gene of tomato (Lycopersicon esculentum Mill.) is required for chloroplast development, palisade cell morphogenesis, and embryogenesis. Previous work suggested that DCL protein is involved in 4.5S rRNA processing. The Arabidopsis thaliana (L.) Heynh. genome contains five sequences encoding for DCL-related proteins. In this paper, we investigate the function of AtDCL protein, which shows the highest amino acid sequence similarity with tomato DCL. AtDCL mRNA was expressed in all tissues examined and a fusion between AtDCL and green fluorescent protein (GFP) was sufficient to target GFP to plastids in vivo, consistent with the localization of AtDCL to chloroplasts. In an effort to clarify the function of AtDCL, transgenic plants with altered expression of this gene were constructed. Deregulation of AtDCL gene expression caused multiple phenotypes such as chlorosis, sterile flowers and abnormal cotyledon development, suggesting that this gene is required in different organs. The processing of the 4.5S rRNA was significantly altered in these transgenic plants, indicating that AtDCL is involved in plastid rRNA maturation. These results suggest that AtDCL is the Arabidopsis ortholog of tomato DCL, and indicate that plastid function is required for normal plant development.Abbreviations DCL Defective chloroplasts and leaves - GFP Green fluorescent protein