LogSpin: a simple, economical and fast method for RNA isolation from infected or healthy plants and other eukaryotic tissues

<p>Abstract</p> <p>Background</p> <p>Rapid RNA extraction is commonly performed with commercial kits, which are very expensive and can involve toxic reagents. Most of these kits can be used with healthy plant tissues, but do not produce consistently high-quality RNA from necrotic fungus-infected tissues or fungal mycelium.</p> <p>Findings</p> <p>We report on the development of a rapid and relatively inexpensive method for total RNA extraction from plants and fungus-infected tissues, as well as from insects and fungi, based on guanidine hydrochloride buffer and common DNA extraction columns originally used for the extraction and purification of plasmids and cosmids.</p> <p>Conclusions</p> <p>The proposed method can be used reproducibly for RNA isolation from a variety of plant species. It can also be used with infected plant tissue and fungal mycelia, which are typically recalcitrant to standard nucleic acid extraction procedures.</p

The Effects of Glucosinolates and Their Breakdown Products on Necrotrophic Fungi

<div><p>Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used <i>Arabidopsis thaliana</i> mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus <i>Botrytis cinerea</i>, which infects a variety of hosts, with the Brassicaceae-specific fungus <i>Alternaria brassicicola</i>. <i>B. cinerea</i> isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while <i>A. brassicicola</i> was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that <i>B. cinerea</i> stimulates the accumulation of glucosinolates to a greater extent than <i>A. brassicicola</i>. In our work with <i>A. brassicicola</i>, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, <i>in vivo</i>, hydrolysis products of indole glucosinolates were found to be involved in defense against <i>B. cinerea</i>, but not in the host response to <i>A. brassicicola</i>. We suggest that the Brassicaceae-specialist <i>A. brassicicola</i> has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist <i>B. cinerea</i> are more sensitive to these phytochemicals.</p></div

Impact of aliphatic glucosinolate on fungal pathogenicity.

<p><i>Arabidopsis</i> leaves from plants containing the double-knockout <i>myb28 myb29</i> (<i>myb28/29</i>) expressed against the Col-0 background (A) and plants in which <i>MYB29^OXP</i> (MYB29) and <i>MYB34^OXP</i> (MYB34) were expressed against the Ler background (B) were inoculated with <i>B. cinerea</i> (B05.10 or grape isolate) or <i>A. brassicicola</i>. Lesion size was measured 72 h after inoculation with <i>B. cinerea</i> and 120 to 192 h after inoculation with <i>A. brassicicola</i>. Average lesion sizes from 10 to 17 leaves of each genotype are presented together with the standard errors for each average. All numbers are presented as the relative lesion size as compared to that observed on the corresponding background wild-type plants. Different letters or asterisks above the columns indicate statistically significant differences at <i>P</i>>0.05, as determined using the Kruskal-Wallis test and Dunn’s test.</p

Effects of glucosinolate-turnover products on fungal pathogenicity.

<p><i>Arabidopsis</i> leaves from wild-type, <i>pen2</i>, <i>cyp81F2</i> and <i>pen2/cyp81F2</i> plants were inoculated with the grape isolate of <i>B. cinerea</i> (upper panel), the B05.10 <i>B. cinerea</i> isolate (middle panel) or <i>A. brassicicola</i> (lower panel). Lesion size was measured 72 h after inoculation with <i>B. cinerea</i> and 120 to 192 h after inoculation with <i>A. brassicicola</i>. Average lesion areas for 30 leaves of each genotype are presented together with the standard error for each average. All numbers are presented as the relative lesion size as compared to the lesions observed on the corresponding background wild-type plants. Different letters above the columns indicate statistically significant differences at <i>P</i><0.05, as determined using the Kruskal-Wallis test and Dunn’s test.</p

Effects of indole glucosinolate and camalexin on fungal pathogenicity.

<p><i>Arabidopsis</i> mutants <i>cyp79B2/B3</i> and <i>pad3</i>, which have altered total glucosinolate and/or camalexin content, and their corresponding wild-type background (Col-0) were inoculated with <i>B. cinerea</i> (B05.10 or grape isolate) or <i>A. brassicicola</i>. Lesion size was measured 72 h after inoculation (upper and middle panels) with <i>B. cinerea</i> and 120 to 192 h after inoculation with <i>A. brassicicola</i> (lower panel). Average lesion sizes from 30 leaves of each genotype are presented along with and the standard error of each average. All numbers are presented as the relative percentage to their corresponding background wild-type. Different letters above the columns indicate statistically significant differences at <i>P</i><0.05, as determined using the Kruskal-Wallis test and Dunn’s test.</p

Effects of glucosinolate-breakdown products on fungal pathogenicity.

<p><i>Arabidopsis</i> mutants with altered total glucosinolate-breakdown product contents and containing different relative amounts of the different type of products were inoculated with the grape isolate of <i>B. cinerea</i> (upper panel), the B05.10 isolate of <i>B. cinerea</i> (middle panel) or <i>A. brassicicola</i> (lower panel). Lesion size was measured 72 h or 120 to 192 h post-inoculation (<i>B. cinerea</i> and <i>A. brassicicola</i>, respectively) on leaves from <i>tgg1-3/tgg2-1</i> (<i>tgg1/2</i>) plants, <i>35S:ESP</i> plants, the wild-types Col-0 and Ler and the triple mutant <i>35:ESP/tgg1-3/tgg2-1</i> (<i>tgg1/2:ESP</i>). (All mutations were expressed against the Col-0 background.) Average lesion areas from 15 to 30 leaves of each genotype are presented together with the standard error of each average. All numbers are presented as the relative lesion size as compared to that observed on the corresponding background wild-type plants. Different letters above the columns indicate statistically significant differences at <i>P</i><0.05, as determined using the Kruskal-Wallis test and Dunn’s test.</p

Glucosinolate accumulation in <i>Arabidopsis</i> after inoculation with a fungal pathogen.

<p>Col-0 <i>Arabidopsis</i> seedlings were inoculated with the B05.10 <i>B. cinerea</i> isolate or <i>A. brassicicola</i> and glucosinolate content was measured 72 h or 120 to 192 h post-inoculation, respectively. GS, glucosinolate. Average glucosinolate accumulation was calculated for 6 to 9 seedlings per treatment and those averages are presented together with their standard errors. Asterisks indicate statistically significant differences relative to the control at <i>P</i><0.05, as indicated by <i>t</i>-tests.</p