Genetic dissection of salicylic acid-mediated defense signaling networks in Arabidopsis

Abstract

Properly coordinated defense signaling networks are critical for the fitness of plants. One hub of the defense networks is centered on salicylic acid (SA), which plays a key role in activating disease resistance in plants. However, while a number of genes are known to affect SA-mediated defense, relatively little is known about how these gene interact genetically with each other. Here we exploited the unique defense-sensitized Arabidopsis mutant accelerated cell death (acd) 6-1 to dissect functional relationships among key components in the SA hub. We show that while enhanced disease susceptibility (eds) 1-2 and phytoalexin deficient (pad) 4-1 suppressed acd6-1-conferred small size, cell death, and defense phenotypes, a combination of these two mutations did not incur additive suppression. This suggests that EDS1 and PAD4 act in the same signaling pathway. To further evaluate genetic interactions among SA regulators, we constructed 10 pairwise crosses in the acd6-1 background among mutants defective in: SA INDUCTION-DEFICIENT 2 for SA biosynthesis; AGD2-LIKE DEFENSE 1, EDS5, and PAD4 for SA accumulation; and NONEXPRESSOR OF PR GENES 1 for SA signaling. Systematic analysis of the triple mutants based on their suppression of acd6-1-conferred phenotypes revealed complex and interactive genetic relationships among the tested SA genes. Our results suggest a more comprehensive view of the gene networks governing SA function and provide a framework for further interrogation of the important roles of SA and possibly other signaling molecules in regulating plant disease resistance.

Figure 1

eds1-2 acts nonadditively with pad4-1 in suppressing acd6-1–conferred phenotypes. (A) Picture of 25-day-old plants. (B) SA quantitation. Free and total SA were extracted from plants shown in A and analyzed by HPLC. (C) Expression of PR1. Total RNA was extracted from uninfected 25-day-old plants for Northern blot analysis. rRNA was used as a loading control. (D) Bacterial growth assay. Plants of 25 days old were infected with PmaDG3 (OD₆₀₀ = 0.0001) and bacterial growth was assessed 3 days after infection. Data represent the average of bacterial numbers in six samples (n = 6) ± SE. In B and D, statistical analysis was performed with Student’s t-test (StatView 5.0.1). Letters indicate significant difference among the samples (P < 0.05). The key for the genotypes used in these experiments is shown in C, right.

Figure 2

eds1-2 acts nonadditively with pad4-1 in suppressing cell death in acd6-1. The fourth to sixth leaves of the indicated genotypes were stained with trypan blue. Photographs were taken with a dissecting microscope connected to an AxioCam MRc5 camera (Zeiss). eds1-2 and pad4-1 showed no detectable cell death (data not shown). Note the large patches of cell death shown in acd6-1 (arrows) were reduced in the double and triple mutants.

Figure 3

Genetic interactions among SA mutants lead to altered acd6-1 morphology. Plants were photographed 25 days postplanting. The single mutants largely resemble Col-0 (data not shown).

Figure 4

Genetic interactions among SA mutants lead to altered SA accumulation in acd6-1. SA was extracted from 25-day-old plants and analyzed by HPLC for free (A) and total SA (B). Note B has a log scale. The single mutants have similar SA levels as Col-0 (data not shown). Letters indicate significant difference among the samples (P < 0.05).

Figure 6

Genetic interactions among SA mutants lead to altered disease resistance in acd6-1. Bacterial growth was assessed 3 days after infection with PmaDG3 (OD₆₀₀ = 0.0001). Statistical analysis was performed with Student's t-test (StatView 5.0.1). Different letters indicate significant difference among the samples (P < 0.05; n = 6).

Figure 7

Genetic interactions among SA mutants lead to altered cell death in acd6-1. The fourth to sixth leaves of the indicated genotypes were stained with trypan blue. Photographs were taken with a dissecting microscope connected to an AxioCam MRc5 camera (Zeiss). The single mutants showed no detectable cell death (data not shown).

Figure 5

Genetic interactions among SA mutants lead to altered PR1 expression in acd6-1. Total RNA was extracted from uninfected 25-day-old plants for northern blot analysis. rRNA was used as a loading control. No PR1 expression was detected in the single mutants, sid2-1, ald1-1, pad4-1, eds5-1, and npr1-1 (data not shown).

Figure 8

SA-mediated defense signaling networks. SA represents a key hub on the defense signaling networks. Genes regulate SA-mediated defense can be viewed in three types. Type I genes are responsible for SA biosynthesis, with SID2 contributing to the major SA production and SID2-independent pathway playing a minor role. Type II genes encode protein products that do not act directly as SA biosynthetic enzymes. It is possible that these SA regulators might influence SA biosynthetic processes by either modifying the activities of the SA biosynthetic enzymes or regulating precursor availability for SA biosynthesis. Alternatively, they can affect SA stability, sequestration, transport, and/or conjugation. Among the known type II SA genes, EDS5 plays a major role in regulating SA accumulation. Other components also partially affect SA levels. Type III genes include NPR1 as the main SA signal transducer and other signal transducers independent of NPR1. Expression of some SA regulatory genes is known to be regulated by SA, suggesting the existence of multiple signal amplification loops involving SA and SA regulatory genes. In this report, we described a genetic analysis based on the defense-sensitized mutant acd6-1 to elucidate the functional relationships among the SA genes. We showed that the same type of SA genes can additively interact with each other and they can also additively interact with other types of SA genes to affect SA accumulation, defense gene expression, disease resistance, and/or cell death phenotypes in the acd6-1 background. Nonadditive interactions were observed between EDS1 and PAD4 and between ALD1 and NPR1.