Silverleaf whitefly induces salicylic acid defenses and suppresses effectual jasmonic acid defenses

Abstract

The basal defenses important in curtailing the development of the phloem-feeding silverleaf whitefly (Bemisia tabaci type B; SLWF) on Arabidopsis (Arabidopsis thaliana) were investigated. Sentinel defense gene RNAs were monitored in SLWF-infested and control plants. Salicylic acid (SA)-responsive gene transcripts accumulated locally (PR1, BGL2, PR5, SID2, EDS5, PAD4) and systemically (PR1, BGL2, PR5) during SLWF nymph feeding. In contrast, jasmonic acid (JA)- and ethylene-dependent RNAs (PDF1.2, VSP1, HEL, THI2.1, FAD3, ERS1, ERF1) were repressed or not modulated in SLWF-infested leaves. To test for a role of SA and JA pathways in basal defense, SLWF development on mutant and transgenic lines that constitutively activate or impair defense pathways was determined. By monitoring the percentage of SLWF nymphs in each instar, we show that mutants that activate SA defenses (cim10) or impair JA defenses (coi1) accelerated SLWF nymphal development. Reciprocally, mutants that activate JA defenses (cev1) or impair SA defenses (npr1, NahG) slowed SLWF nymphal development. Furthermore, when npr1 plants, which do not activate downstream SA defenses, were treated with methyl jasmonate, a dramatic delay in nymph development was observed. Collectively, these results showed that SLWF-repressed, JA-regulated defenses were associated with basal defense to the SLWF.

Figure 1.

Summary of Arabidopsis defense gene expression patterns after SLWF second and third instar feeding. Genes involved in SA- and JA/ET-defense signaling and SA and JA biosynthesis pathways are shown as colored boxes. Green and red denote an increase or decrease in RNA levels, respectively (<1.5-fold). Blue indicates no change in expression. Several ethylene receptor genes have been identified, including ETR1, ERS1, EIN4, ERS2, and ETR2; only ETR1 and ERS1 are illustrated. These gene expression trends are based on microarray studies reported by Kempema et al. (2007).

Figure 2.

Defense gene transcript accumulation after infestation with SLWF nymphs. Total RNA was extracted from SLWF-infested or control noninfested rosette leaves. Infestations were performed at 22°C. cDNAs were synthesized and used in PCR reactions with gene-specific primers for defense genes involved in SA-dependent pathway (SID2, EDS5, PAD4, PR1, BGL2, PR5) and JA/ET-dependent pathway (ERS1, ERF1, THI2.1, VSP1, PDF1.2, FAD3). ACTIN7 was used as a control (20 cycles). A, RT-PCRs were performed on RNAs isolated from SLWF-infested and noninfested plant leaves collected at 0, 7, 14, 21, and 28 d. SA- and JA-regulated gene RNAs were detected after 25 cycles of PCR. B, Leaves from 21-d infested (I) and control noninfested (C) plants were collected. SA- and JA-regulated gene RNAs were detected after 25 and 27 cycles of PCR, respectively. RT-PCRs were performed on the RNAs used in the microarray experiments (Kempema et al., 2007) and RNAs from two additional infestation experiments (see “Materials and Methods”). A representative experiment is displayed.

Figure 3.

Local and systemic accumulation of SA- and JA-defense gene RNAs. Infested leaves (local) and noninfested, apical leaves (systemic) from 21-d SLWF-infested plants (I) were collected. Control tissue (C) was collected from developmentally matched leaves on noninfested plants. PCR was performed on cDNA using gene-specific primers (25 cycles for SA-regulated genes and 27 cycles for JA-regulated genes). ACTIN7 was used as a control (20 cycles). Three biological replicate infestations at 22°C were performed. One representative experiment is shown.

Figure 4.

SLWF nymph development on mutant, transgenic, and wild-type plants. At 24 d postinfestation, the numbers of total nymphs and nymphs in their first (gray), second (dotted), third (white), and fourth (black) instars were counted and the percentage of insects in each instar on wild-type Columbia (WT) and SA- and JA-signaling mutant/transgenic lines determined. Defense signal mutants and lines are described within “Results” and include the following: SA-deficient (npr1-1, NahG), JA-deficient (coi1-1), SA overexpression (cim10), and JA overexpression (cev1) plant lines. Three biological replicate infestations were performed at 22°C and analyzed, and a representative experiment is shown. The infestation level and biological variation within the replicate plants of each genotype can be found in Supplemental Figure S1. Significance of variation in percentage of fourth instars across genotypes was determined using Tukey's multiple comparison analysis at the 99.6% individual confidence level. The significance is indicated by the following: a, b, or c. On average, infested plants had approximately 107 nymphs.

Figure 5.

Local accumulation of defense gene RNAs. RNAs for sentinel SA (PR1, BGL2) and JA (VSP1, PDF1.2) defense-response genes were monitored by RT-PCR using gene-specific primers and 25 and 27 cycles, respectively. cDNAs were synthesized from RNAs from leaves from uninfested (C) mutant and control plants or 24-d infested leaves (I) from mutant and control plants. ACTIN7 was used as a control (20 cycles). Each infested plant had approximately 107 feeding nymphs. Three biological replicate infestations at 22°C were performed. One representative experiment is shown.

Figure 6.

SLWF development on MeJA-treated or control npr1-1 plants. At 17 d postinfestation, the numbers of nymphs in each instar were counted on npr1-1 plants treated with 100 μm MeJA/0.001% ethanol (diagonal bars) or 0.001% ethanol (black bars; control). The percentage of nymphs at each developmental stage (first through fourth instar) was determined. Infestations were performed at 24°C, which accelerated SLWF nymph development. Each infested plant had approximately 225 feeding nymphs. Two biological replicate infestations were performed. One representative experiment is shown.