The false smut pathogen Ustilaginoidea virens requires rice stamens for false smut ball formation.

Rice false smut has emerged as a serious grain disease in rice production worldwide. The disease is characterized by the transformation of individual rice florets into false smut balls, which is caused by the fungal pathogen Ustilaginoidea virens. To date, little is known about the host factors required for false smut ball formation by U. virens. In this study, we identified histological determinants for the formation of false smut balls by inoculating U. virens into rice floral mutants defective with respect to individual floral parts. The results showed that U. virens could form mature false smut balls in rice floral mutants with defective pistils, but failed to develop false smut balls in the superwoman mutant lacking stamens, identifying that U. virens requires rice stamens to complete its infection cycle. Comparative transcriptome analysis indicated a list of candidate host genes that may facilitate nutrient acquisition by U. virens from the rice stamens, such as SWEET11, SWEET14 and SUT5, and genes involved in the biosynthesis of trehalose and raffinose family sugars. These data pinpoint rice stamens as the key target organ of U. virens infection and provide a valuable starting point for dissecting the molecular mechanism of false smut ball formation.

[Ultrastructural observation related to cell-to-cell movement and long-distance systemic transport on the hosts infected with BBWV 2].

The alteration of ultrastructure in Pisum sativum and Vicia faba leaf cells infected with B935 isolate of BBWV 2 were investigated by electron microscopy, immunogold-labeling technique. The results showed that the membranous proliferation, virus-formed crystals and tubular structures were found in leaf cells of two hosts. At early stages of infection, the tubules containing virus-like particles associate with plasmodesmata in mesophyll cell. Immunogold particles anti-BBWV 2 were localized to the plasmodesmata modified by tubules passing through them. The membranous proliferation and virus-formed tubules were also found in the parenchyma cells, companion cells and transfer cells of vascular bundle. Some virus-like particles located within sieve tube can be labeled immunogold particles anti-BBWV 2. These suggest that BBWV 2, similar CPMV, produce tubules extending into the plasmodesmata. Virions assembled in the cytoplasm are escorted to the tubular structures through interactions with their MP and are then transported to the adjacent cell. Many 160 nm in diameter virus-formed tubules in the cytoplasm, as a special aggregate, not directly relate to cell-to-cell movement; Intact virions are long-distance sustemic transported possibly through sieve elements.

Villosiclava virens infects specifically rice and barley stamen filaments due to the unique host cell walls.

Rice false smut, caused by the fungal pathogen Villosiclava virens, is one of the most important rice diseases in the world. Previous studies reported that the pathogen has less number of cell wall-degraded genes and attacks dominantly rice stamen filaments and extends intercellularly. To reveal why the fungus infects plant stamen filaments, inoculation test on barley was carried out with the similar protocol to rice. The experimental results showed that the fungus could penetrate quickly into barley stamen filaments and extends both intracellularly and intercellularly, usually resulting in severe damage of the stamen filament tissues. It also attacked young barley lodicules and grew intercellularly by chance. The light microscopic observations found that the epidermal and cortex cells in barley stamen filaments arranged loosely with very thick cell walls and large cell gaps. Cellulose microfibrils in barley stamen filament cell walls arranged very sparsely so that the cell walls looked like transparent. The cell walls were very soft and flexible, and often folded. However, V. virens extended dominantly in the noncellulose regions and seemed never to degrade microfibrils in barley and rice cell walls. This suggested that the unique structures of rice and barley stamen filaments should be fit for their function of elongation in anthesis, and also endow with the susceptibility to the fungus, V. virens.

Expression of a begomoviral DNAbeta gene in transgenic Nicotiana plants induced abnormal cell division.

An increasing number of monopartite begomoviruses are being identified that a satellite molecule (DNAbeta) is required to induce typical symptoms in host plants. DNAbeta encodes a single gene (termed betaC1) encoded in the complementary-sense. We have produced transgenic Nicotiana benthamiana and N. tabacum plants expressing the betaC1 gene of a DNAbeta associated with Tomato yellow leaf curl China virus (TYLCCNV), under the control of the Cauliflower mosaic virus 35S promoter. Transgenic plants expressing betaC1 showed severe developmental abnormalities in both species. Microscopic analysis of sections of both transgenic and non-transgenic N. tabacum leaves showed abnormal outgrowths of transgenic N. tabacum to be due to disorganized cell division (hyperplasia) of spongy and palisade parenchyma. Immuno-gold labeling of sections with a polyclonal antibody against the betaC1 protein showed that the betaC1 protein accumulated in the nuclei of cells. The possible biological function of the betaC1 protein was discussed.

[The immunogold localization of Rubisco and its activase in chloroplasts of barley and maize leaves].

Cellular localization of Rubisco and Rubisco activase (RCA) in the C(3) plant barley (Hordeum vulgare L.) and the C(4) plant maize (Zea mays L.) leaves was investigated using immunogold-labeling electron microscopy. The results showed that the leaf anatomy and the immunolocalization of the two photosynthetic enzymes were markedly different between barley and maize. In barley, the mesophyll chloroplasts had well-developed grana and their stroma was densely labeled with immunogold particles for Rubisco and for RCA. In maize, the mesophyll chloroplasts had well-developed grana but their stroma was scarcely labeled with immunogold particles, indicating a low Rubisco content. But the chloroplasts of the bundle sheath cells had only few rudimentary grana and their stroma was densely labeled with immunogold particles for Rubisco. A higher density of immunogold particles for RCA was located both in chloroplast stroma of the bundle sheath cells and the mesophyll cells. These results showed that the structure and function of chloroplasts are different between C(3) plants and C(4) plants.

Identification of Colletotrichum spp. isolated from strawberry in Zhejiang Province and Shanghai City, China.

Strawberry anthracnose, caused by Colletotrichum spp., is a major disease of cultivated strawberry. This study identifies 31 isolates of Colletotrichum spp. which cause strawberry anthracnose in Zhejiang Province and Shanghai City, China. Eleven isolates were identified as C. acutatum, 10 as C. gloeosporioides and 10 as C. fragariae based on morphological characteristics, phylogenetic and sequence analyses. Species-specific polymerase chain reaction (PCR) and enzyme digestion further confirmed the identification of the Colletotrichum spp., demonstrating that these three species are currently the causal agents of strawberry anthracnose in the studied regions. Based on analysis of rDNA internal transcribed spacers (ITS) sequences, sequences of all C. acutatum were identical, and little genetic variability was observed between C. fragariae and C. gloeosporioides. However, the conservative nature of the MvnI specific site from isolates of C. gloeosporioides was confirmed, and this site could be used to differentiate C. gloeosporioides from C. fragariae.

[Prokaryotic expression, antiserum preparation and cytochemical analysis of TaMlo3 in wheat].

Mlo gene in wheat is critical to determine wheat broad spectrum disease resistance against the pathogenic powdery mildew fungus. According to wheat TaMlo3 cDNA sequence, the two DNA fragments encoding the intracellular loop 2 and C-terminus of wheat Mlo protein were cloned respectively into the vector pET-30a, and then the fusion polypeptides were expressed in E. coli BL21. The recombinant proteins were purified by Ni2+ -NTA agarose column, and then were used to immunize the rabbits respectively. Two polyclonal antisera were obtained and they could be specifically bound on the extrahaustorial membrane and extrahaustorial matrix of the powdery mildew fungus by immuno-gold labeling.

[Distribution of Rubisco and RCA in Brassica chinensis chloroplasts and effect of TuMV-infection on their cellular localization].

The cellular localizations of Rubisco and Rubisco activase (RCA) in Chinese cabbage (Brassica chinensis L. cv. Suzhou) leaves were investigated by immunogold-labeling electron microscopy. The results showed that Rubisco and RCA were mainly located in chloroplasts of mesophyll, guard cell of stomatal apparatus and parenchyma of vascular bundle. A high density of gold particles were localized preferentially to the chloroplast stroma. In contrast, there were no specific binding of gold particles detected in the cytoplasm, vacuole, mitochondria and other organelles. As infected by turnip mosaic virus, the density of gold particles decreased lightly in the abnormal chloroplasts and dropped by much lower (58.44% and 64.67% as that in the health chloroplasts respectively) in swollen chloroplasts. This indicated that virus infection caused the decreases of Rubisco and RCA in host plant, which then affected plant photosynthesis.

[Activity assay, antiserum preparation and cellular localization of ustiloxins].

False smut is an increasingly important rice disease in recent years, caused by a pathogen, Ustilaginoidea virens Cooke, Takahashi. The pathogen fungus grew well in PD medium, and could produce toxins, ustiloxins, which strongly inhibited the growth of radicle and embryo of plants. The rough ustiloxins were extracted with 100% methanol from the culture filtrate in the study. The antiserum against Ustiloxin A, the main component of the toxins, was raised by immunizing New Zealand rabbit. Elisa assay showed that the titers of the two antiserums obtained were 1: 20 000 and 1: 6 000, respectively. The specificity of the antiserums was also confirmed by Immuno-gold labeling.