Transgenic Arabidopsis thaliana containing increased levels of ATP and sucrose is more susceptible to Pseudomonas syringae

Abstract

Disease resistance exerts a fitness cost on plants, presumably due to the extra consumption of energy and carbon. In this study, we examined whether transgenic Arabidopsis thaliana with increased levels of ATP and sucrose is more resistant or susceptible to pathogen infection. Lines of A. thaliana over-expressing purple acid phosphatase 2 (AtPAP2) (OE lines) contain increased levels of ATP and sucrose, with improved growth rate and seed production. Compared to wild type (WT) and pap2 lines, the OE lines were more susceptible to several Pseudomonas syringae pv. tomato (Pst) strains carrying AvrRpm1, AvrRpt2 AvrRps4, AvrPtoB, HrcC and WT strain DC3000. The increased susceptibility of the OE lines to Pst strains cannot solely be attributed to the suppressed expression of R-genes but must also be attributed to the suppression of downstream signaling components, such as MOS2, EDS1 and EDS5. Before infection, the levels of salicylic acid (SA) and jasmonic acid (JA) precursor OPDA were similar in the leaves of OE, pap2 and WT plants, whereas the levels of JA and its derivative JA-Ile were significantly lower in the leaves of OE lines and higher in the pap2 line. The expression of JA marker defense gene PDF1.2 was up-regulated in the OE lines compared to the WT prior to Pst DC3000 infection, but its expression was lower in the OE lines after infection. In summary, high fitness Arabidopsis thaliana exhibited altered JA metabolism and broad suppression of R-genes and downstream genes as well as a higher susceptibility to Pst infections.

Fig 1. AtPAP2 OE Arabidopsis thaliana lines display enhanced susceptibility to Pseudomonas syringae pv. tomato (Pst) strains.

(a) Phenotypes of WT, pap2, OE7 and OE21 A. thaliana lines at 5 dpi with Pst DC3000strains carrying different Avr genes in greenhouse conditions (16/8 hr light/dark). (b) Phenotype of WT, pap2, OE7 and OE21 A. thaliana lines at 5d pi with Pst DC3000 in growth chamber conditions (10/14 hr light/dark). We inoculated 25 day-day-old plants (greenhouse) and 30-day-old plants (growth chamber).

Fig 2. Biomass quantification of the Pseudomonas syringae pv. tomato DC3000 strains (Pst).

DNA was extracted from inoculated plants grown in greenhouse conditions (16/8 hr light/dark) at 5 dpi, and the bacterial biomass was quantified using qPCR on the oprF gene. Bacterial biomass was normalized to the amplicon of AtRuBisCO, and its amount in the WT plants was normalized as 1 in each panel. DC3000: (Pst) strain DC3000; AvrRpm1: Pst DC3000 carrying AvrRpm1; AvrRpt2: Pst DC3000 carrying AvrRpt2, AvrRps4: Pst DC3000 carrying AvrRps4; AvrPtoB: Pst DC3000 carrying AvrPtoB (*P< 0.01).

Fig 3. Recovery of infected plants after inoculation with P. syringae AvrRps4.

Photos were taken from 58-day-old plants grown under short day (10/14 hr light/dark photoperiod).

Fig 4. Sensitivity of Arabidopsis thaliana seeds of WT, pap2 and overexpression lines OE7 and OE21 to jasmonic acid (JA) and benzoic acid (BA).

Seeds of OE lines along with pap2 and WT were germinated on MS or MS supplemented with 5 μM JA or 5 μM BA for 12 days. Note that OE lines are more resistant to JA treatment than WT, whereas the pap2 line was more susceptible than the WT.

Fig 5. SA and JA levels in leaves of 3-week-old Arabidopsis thaliana plants.

Leaves were collected from 3-week-old WT (Col-0), pap2 mutant and OE lines (OE-7 and OE-21) plants. SA, JA, JA conjugated with isoleucine (JA-Ile) and JA precursor 12-oxo-phytodienoic acid (OPDA) levels were determined by LC/MS. D6-SA and D5-JA were added as internal standards. *P< 0.05; **P< 0.01 by Student’s t-test.

Fig 6. Relative expression of SA and JA defense pathway marker genes PR1 and PDF1.2, respectively, in the four lines (WT, pap2, OE7 and OE21) were determined by qRT-PCR at three time points (t = 0 hpi, t = 24 hpi and t = 48 hpi) after Pseudomonas syringae pv. tomato DC3000 inoculation.

Samples were collected in three biological replicates (**P< 0.01), and the expression level in WT at each time point was set to 1 1 for normalization.