Characterization of the interaction between the dark septate fungus Phialocephala fortinii and Asparagus officinalis roots.

Phialocephala fortinii Wang & Wilcox is a member of root-inhabiting fungi known collectively as dark septate endophytes (DSE). Although very common and distributed worldwide, few studies have documented their interaction with roots on a structural basis. The objective of this study was to determine the early colonization events and formation of microsclerotia of P. fortinii in roots of Asparagus officinalis L., a species known to have DSE. A loose network of hyphae accumulated at the root surface, and coils formed around root hairs and external to epidermal cells overlying short cells of the dimorphic, suberized exodermis. Root penetration occurred via swollen, appressorium-like structures into epidermal cells where coiling of hyphae occurred along the periphery of the cells. Hyphae penetrated from the epidermis into short exodermal cells and from these into cortical cells. Hyphae colonized the cortex up to the endodermis and sometimes entered the vascular cylinder. Some root tips were colonized as well. Microsclerotia in epidermal and exodermal short cells accumulated glycogen, protein, and polyphosphate. Energy-dispersive X-ray spectroscopy on distinct bodies visible in microsclerotial hyphae revealed high levels of phosphorus.

Improved RNA extraction and one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in plant extracts.

A procedure was developed for simultaneous detection of plant RNA viruses and of plant RNA, as a control. RT-PCR amplification with primers designed for the detection of the plant mRNAs encoding malate dehydrogenase (MDH) and the large subunit of ribulose bisphosphate carboxylase oxygenase (RubiscoL) was used for the development of a plant extraction procedure that consistently yields extracts that can be amplified. The control amplification was used successfully on extracts from cane, leaf and/or bud tissues from grapevine, apple, raspberry, strawberry, peach, apricot, plum and wheat. Multiplex RT-PCR conditions were established for the simultaneous detection in grapevine extracts of either arabis mosaic virus, rupestris stem pitting associated virus and malate dehydrogenase mRNA, or grapevine virus A, grapevine virus B, grapevine leafroll associated virus-3, and RubiscoL mRNA.

Inhibition of alfalfa mosaic virus RNA and protein synthesis by actinomycin D and cycloheximide.

Actinomycin D, added early after inoculation, reduces the production of infectious alfalfa mosaic virus in cowpea protoplasts by 90%. This reduction was associated with an inhibition of viral minus-strand and plus-strand RNA synthesis, suggesting the involvement of host factors in these processes. Coat protein production was less affected by the drug. Addition of cycloheximide throughout the growth cycle resulted in an immediate cessation of coat protein production and an enhanced degradation of viral RNA. This degradation obscured possible effects of the drug on viral RNA synthesis.

Replication of temperature-sensitive mutants of alfalfa mosaic virus in protoplasts.

Six mutants of alfalfa mosaic virus (AIMV), previously found to have conditionally lethal defects when inoculated to tobacco leaf discs, were assayed for a temperature-sensitive (ts) phenotype in cowpea protoplasts. At 30 degrees the virus production of four mutants was less than 10% of that obtained at 25 degrees , whereas wild-type AIMV multiplied with similar efficiencies at both temperatures. Supplementation experiments performed in protoplasts with two mutants indicated the presence of mutations on both RNA 1 and RNA 2. Viral plus- and minus-strand RNA synthesis induced by these mutants was monitored by the Northern blotting technique under various supplementation conditions. The ts defect was mainly in the production of viral minus-strand RNA whereas transcription of minus-strand into genomic and subgenomic RNAs was much less affected. In addition, results indicate that replication of RNA 1 requires a function not essential for the synthesis of the other RNAs. One of the mutants showed a host-dependent expression of ts mutations, probably reflecting that host-viral interactions play an important role in A1MV replication.

Time course of alfalfa mosaic virus RNA and coat protein synthesis in cowpea protoplasts.

A study was made of the time course of the synthesis of viral plus-strand RNA, minus-strand RNA, and coat protein in alfalfa mosaic virus-infected Cowpea protoplasts. The three genomic RNAs were synthesized at different rates, as were their corresponding minus-strands. We conclude that viral RNA synthesis is regulated both at the level of minus-strand production and the level of plus-strand production. The synthesis of subgenomic RNA 4 was slower than that of its corresponding genomic RNA (RNA 3), indicating that an additional function, expressed later in infection, is required for production of subgenomic coat protein messenger. The data support a model for RNA 4 synthesis involving internal initiation by the RNA polymerase at the intercistronic junction in minus-strand RNA 3. The temporal relationship of the synthesis of RNA 3, RNA 4, and coat protein is discussed.

Altered balance of the synthesis of plus- and minus-strand RNAs induced by RNAs 1 and 2 of alfalfa mosaic virus in the absence of RNA 3.

The synthesis of viral plus-strand and minus-strand RNAs in cowpea protoplasts inoculated with mixtures of alfalfa mosaic virus nucleoproteins (B, M, Tb, and Ta) was analyzed by the Northern blotting technique. A mixture of B, M, and Tb induced the synthesis of plus-strand RNAs 1, 2, 3, and 4 and three minus-strand RNAs corresponding to RNAs 1, 2, and 3, respectively. Compared to this complete infection, a mixture of B and M induced the synthesis of a reduced amount of plus-strand RNAs 1 and 2 and a greatly enhanced amount of minus-strand RNAs 1 and 2. No detectable viral RNA synthesis was induced by mixtures of B and Tb or M and Tb. It is concluded that expression of genomic RNAs 1 and 2 results in the formation of a replicase activity that produces roughly equal amounts of viral plus- and minus-strand RNAs and that an RNA 3-encoded product, possibly the coat protein, is responsible for a switch to an asymmetric production of viral plus-strand RNA. The observation that no minus-strand corresponding to the subgenomic RNA 4 is produced suggests that recognition of the genome segments by the viral replicase involves sequences outside the 3'-terminal regions that are homologous to RNA 4.