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. 2002 May;129(1):169-80.
doi: 10.1104/pp.010940.

A plant gene up-regulated at rust infection sites

Michael A Ayliffe  1 , James K Roberts, Heidi J Mitchell, Ren Zhang, Gregory J Lawrence, Jeffrey G Ellis, Tony J Pryor
Affiliations

A plant gene up-regulated at rust infection sites

Michael A Ayliffe et al. Plant Physiol. 2002 May.

Abstract

Expression of the fis1 gene from flax (Linum usitatissimum) is induced by a compatible rust (Melampsora lini) infection. Infection of transgenic plants containing a beta-glucuronidase (GUS) reporter gene under the control of the fis1 promoter showed that induction is highly localized to those leaf mesophyll cells within and immediately surrounding rust infection sites. The level of induction reflects the extent of fungal growth. In a strong resistance reaction, such as the hypersensitive fleck mediated by the L6 resistance gene, there is very little fungal growth and a microscopic level of GUS expression. Partially resistant flax leaves show levels of GUS expression that were intermediate to the level observed in the fully susceptible infection. Sequence and deletion analysis using both transient Agrobacterium tumefaciens expression and stable transformation assays have shown that the rust-inducible fis1 promoter is contained within a 580-bp fragment. Homologs of fis1 were identified in expressed sequence tag databases of a range of plant species including dicots, monocots, and a gymnosperm. Homologous genes isolated from maize (Zea mays; mis1), barley (Hordeum vulgare; bis1), wheat (Triticum aestivum; wis1), and Arabidopsis encode proteins that are highly similar (76%-82%) to the FIS1 protein. The Arabidopsis homologue has been reported to encode a delta1-pyrroline-5-carboxylate dehydrogenase that is involved in the catabolism of proline to glutamate. RNA-blot analysis showed that mis1 in maize and the bis1 homolog in barley are both up-regulated by a compatible infection with the corresponding species-specific rust. The rust-induced genes homologous to fis1 are present in many plants. The promoters of these genes have potential roles for the engineering of synthetic rust resistance genes by targeting transgene expression to the sites of rust infection.

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Figures

Figure 1
Figure 1
Design of gene constructs used for flax transformation. Construct 1, fis1-2.5P-GUS consists of a 2.5-kb DNA fragment that contains the fis1 promoter and 5′-untranslated region (white box underlined with an arrow), in addition to the first 33 codons of the fis1 open reading frame (ORF; horizontally hatched box) fused in frame to a GUS reporter gene (obtuse hatched box). Also encoded on this 2.5-kb fragment is a tRNA-Ala gene depicted as a black box and described in the text. An Agrobacterium tumefaciens nopaline synthase (nos) 3′ transcription terminator is present at the 3′ end of this construct (checkered box). Construct 2, fis1-0.6P-GUS differs from fis1-2.5P-GUS in that 1,889 bp of extreme 5′ sequence has been deleted from the 2.5-kb fragment encoding the fis1 promoter. This construct uses the first 580 bp of sequence immediately upstream of the translation initiation site of the fis1 ORF for expression. On each map, numbering refers to the distance in bp from the translation start site of the fis1 ORF.
Figure 2
Figure 2
Phenotypes of rust-infected fis1-GUS transgenic flax plants. A, Expression of the fis1-2.5P-GUS transgene in flax plants infected with a compatible race of rust. Both leaves have been infected with flax rust for 6 d, stained for GUS activity, and cleared. The upper leaf is derived from a nontransgenic control plant, whereas the lower leaf is from a flax plant containing the fis1-2.5P-GUS transgene. Each GUS-staining spot on the lower leaf corresponds precisely to a rust infection site. Small brown dots visible on the upper leaf were developing rust pustules. B through F, Expression of the fis1-2.5P-GUS transgene in flax plants during a highly incompatible rust infection. B, Leaf from a flax plant containing an fis1-2.5P-GUS transgene and an endogenous L6 resistance gene, 8 d after infection with rust strain CH5 that contains the corresponding A-L6 avirulence gene. A second rust infected leaf from the same plant was GUS stained (C) and only a small amount of GUS expression was detected around infection sites (D). The boxed region in C is enlarged in D. As a control, the same plant line was infected with a fully compatible rust race (E) and abundant GUS expression was detected around infection sites (F). G and H, Expression of the fis1-2.5P-GUS transgene during an intermediate resistance response. G, Leaf from a flax plant containing an fis1-2.5P-GUS transgene and an endogenous P3 resistance gene, 8 d after infection with rust strain CH5 which contains the corresponding A-P3 avirulence gene. A second rust infected leaf from this plant was GUS stained and is shown in H. I, Two flax seedlings GUS stained and cleared, 3 d after infiltration with an A. tumefaciens strain containing a GUS reporter gene under the control of a 35S promoter. An intron was incorporated into the GUS gene to prevent A. tumefaciens expression of a functional GUS protein. J, Flax leaves that had been infected with flax rust for 5 d were vacuum infiltrated with an A. tumefaciens strain containing the fis1-2.5P-GUS transgene. Three days postinfiltration leaves were stained for GUS activity and cleared. GUS activity is observed exclusively around rust infection sites and orange rust pustules can be seen inside these GUS-staining regions.
Figure 3
Figure 3
Structure of the mis1 and AtP5CDH genes. A, Schematic diagram (not to scale) of the mis1 gene and flanking sequence. The 15 exons of the mis1 gene (transcribed from left to right) are represented as gray boxes with the polyadenylation site positions indicated with a vertical arrow. Upstream of the mis1 gene is part of a second predicted gene encoding a maize Rab7 homolog, labeled Rab7h. The first five exons of the predicted Rab7h gene are shown as black boxes with the direction of transcription shown as a bent arrow. The small line beneath the figure delineates the 434-bp RT-PCR fragment from the mis1 gene (excluding the intervening intron) used as a probe in Figures 4 and 6. Horizontal arrows indicate distances in kb. B, Schematic diagram of the AtP5CDH gene and flanking regions. The 16 exons of the gene, which are transcribed from left to right, are shown as gray boxes. A second gene encoding a 16-kD ubiquitin-conjugating enzyme (16 kD uce) is located 843 bp 5′ of the first exon of the gene. The first exon and part of the second exon of this gene are shown as black boxes. The direction of transcription of the ubiquitin conjugating enzyme gene is shown as a bent arrow. Horizontal arrows indicate distances in kb and bp. Numbers beneath the figure refer to the nucleotide position of this sequence on Arabidopsis bacterial artificial chromosome (BAC) clone K19B1 (GenBank accession no. AB015469).
Figure 4
Figure 4
Grass species contain sequences homologous to mis1. DNA blot showing hybridization of the 434-bp mis1 probe to NcoI-digested total DNA isolated from pearl millet (Pennisetum glaucum), panicum (Panicum miliaceum), maize, sugarcane (Saccharum officinarum), barley, wheat, oats (Avena sativa), Aegilops tauschii, sorghum (Sorghum bicolor), and rice (Oryza sativa; lanes 1–10, respectively). Mr sizes are indicated on the figure.
Figure 5
Figure 5
A PileUp sequence alignment of the predicted BIS1, WIS1, MIS1, AtP5CDH, and FIS1 proteins. Accession numbers for these sequences are as follows: AAL70106, AAL70109, AAL70108, AAK73756, and CAA60412, respectively. The AtP5CDH protein is labeled ′aris1′ in this alignment. Identical amino acids are shaded in black, whereas similar amino acids are highlighted in gray. Numbers on the right hand side indicate amino acid positions.
Figure 6
Figure 6
Induction of the maize mis1 and barley bis1 genes during rust infection. A, RNA-blot analysis of maize mis1 gene expression during maize rust infection. Lanes 1 through 4, 5 through 8, and 9 through 12 contain total leaf RNA isolated from three maize lines containing the Rp1-D, Rp5, and Rp1-M rust resistance genes, respectively. Lanes 1, 5, and 9, labeled C, contain RNA isolated from maize plants that had not been infected with maize rust. Lanes 3, 4, 8, and 10, labeled S, contain RNA from each maize line after infection with a compatible race of maize rust, whereas lanes 2, 6, 7, 11, and 12, labeled R, contain RNA from each maize line after infection with an incompatible rust race. A 434-bp PCR product amplified from the mis1 gene was used as a hybridization probe. Beneath the autoradiograph is shown the RNA formaldehyde gel that was subsequently transferred for the RNA-blot analysis. B, RNA-blot analysis of barley bis1 gene expression during infection with a virulent race of Puccinia graminis f.sp tritici. Total RNA was isolated from barley cv Golden Promise (lanes 1–5) and cv Morex (lanes 6–10) at 0 (lanes 1 and 6), 2 (lanes 2 and 7), 4 (lanes 3 and 8), 6 (lanes 4 and 9), and 8 (lanes 5 and 10) d post rust inoculation. The same probe as in A was used for hybridization. Shown beneath the autoradiograph is the RNA gel that was transferred for RNA-blot analysis.
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