Gene expression analysis of Nadefensin in WT and transgenic N. attenuata plants silenced for Nadefensin
In N. attenuata, Nadefensin (NCBI accession AY456268) is up-regulated in WT N. attenuata plants after attack from M. sexta [14,15], Tupiocorus notatus, Myzus nicotianae, Spodoptera littoralis and Trichoplusia ni  larvae. Bacteria (Pseudomonas syringae) are also known to induce defensin in different plants [17,18]. Recently, we reported that Nadefensin was up-regulated 12 h after WT N. attenuata plants were infected with PST DC3000 ; moreover, silencing Nadefensin by RNAi by stable transformation (irdefensin lines 76 and 96) increased the plant's susceptibility to PST DC3000 .
While attack from both M. sexta larvae and PST DC3000 is known to elicit Nadefensin transcripts and protein in N. attenuata, the relative responses to M. sexta larvae and PST DC3000 challenges were not known. We re-examined the levels of Nadefensin transcripts accumulation in PST DC3000 and M. sexta-attacked
plants at a single time point (4 days after pathogen and herbivore
damage). The quantitative real-time PCR (qRT-PCR) analysis (Fig. 1) revealed that Nadefensin transcript accumulation differed significantly across treatments and genotypes (Fig. 1; ANOVA F11,17 = 16.00, P < 0.001): Nadefensin levels in WT plants infected with PST DC3000 and in those attacked by M. sexta did not differ significantly (Fig. 1; p = 0.183). Consistent with our earlier observation , WT plants either damaged by M. sexta or infected by PST DC3000 had significantly more (at least 60%) Nadefensin transcripts compared to similarly treated irdefensin plants (76 and 96). The similar levels of Nadefensin after M. sexta damage or PST DC3000 infection suggest that Nadefensin is likely elicited by jasmonates which are produced in response to pathogen infection as well as insect attack .
Figure 1. M. sexta damage and Pst DC3000 inoculation increase Nadefensin transcripts; responses are highly attenuated in irdefensin (76 and 96) lines. Quantitative real-time PCR (qRT-PCR) was used to analyze Nadefensin transcript accumulation in WT N. attenuata plants and irdefensin (76 and 96) lines in response to continuous M. sexta feeding by first-instar larvae for 4 days or inoculation with Pseudomonas syringae pv tomato DC3000 (Pst DC3000) (1 × 105 cells/ml). Values are mean (± SE) Nadefensin transcripts from 3 replicate plants per treatment normalized to the transcript abundance of actin, which is unregulated under these conditions. Different letters indicate significant differences between genotypes damaged by M. sexta and infected by Pst DC3000.
Effects of PST DC3000 infection and Nadefensin silencing on herbivore performance
Since Nadefensin is expressed in response to attack from both herbivores and pathogens, we asked if silencing Nadefensin expression influenced M. sexta growth in uninduced plants as well as in plants previously inoculated with PST DC3000. We carried out assays on WT and Nadefensin-silenced
plants (lines 76 and 96) which were either uninduced or had been
infected (4 days earlier) with PST DC3000. We measured two parameters
that reflect the overall performance of M. sexta larvae: percentage of leaf area damage and larval mass gain.
Percentage of leaf area damage
After 12 days of attack from a single M. sexta, leaves were evaluated for the percentage of leaf area damaged. M. sexta larvae removed significantly more leaf area from uninduced WT and irdefensin (76 and 96) plants (at least 30%) than from PST DC3000-infected plants (Fig. 2A and 2B; ANOVA, F5,88 = 19.67, P < 0.001). Within the uninduced treatment, no significant differences in the percentage of leaf area damage between WT and irdefensin line 76 (p = 0.905) plants or between WT and irdefensin line 96 (p = 0.517) plants were observed (Fig. 2A).
On the other hand, prior infection with PST DC3000 resulted in greater
leaf area losses (at least 35%) in WT plants compared to plants from
both irdefensin lines (Fig. 2A and 2B; line 76 p < 0.001; line 96 p = 0.002).
Figure 2. Pst DC3000 inoculation and Nadefensin silencing decreases leaf area damage by M. sexta larvae in N. attenuata. A) Mean (± SE) percentage of leaf area damage by M. sexta larvae on WT plants and irdefensin lines
76 and 96. A neonate larva was placed in a clip cage and allowed to
feed for 12 days before the percentage of leaf area damage was
estimated. B) Photographs taken after 12 days of M. sexta feeding on WT and irdefensin lines
76 and 96 that were either uninduced (left) or inoculated with Pst
DC3000 (right). Different letters indicate significant differences
between treatments and genotypes (N = 18).
Larval mass gain
We also measured the mass of the larvae that fed on uninduced and on PST DC3000-infected WT and irdefensin (76 and 96) plants. ANOVA revealed significant differences among the treatments and the genotypes (Fig. 3A and 3B; ANOVA, F17,426 = 14.14, P <
0.001), but the larval mass differences differed from those of the
pattern leaf area damaged. No significant differences in larval mass
between the M. sexta larvae that fed on the uninduced WT and those that fed on WT plants which were PST DC3000 infected was observed (p = 0.264). Within the uninduced treatment, no statistical differences in the mass of larvae that fed on WT and irdefensin line 76 (p = 0.427) plants or WT and irdefensin line 96 (p = 0.117) plants were observed (Fig. 3A).
On the other hand, larvae that fed on WT plants infected with PST
DC3000 gained significantly more (at least 70%) mass than did larvae
that fed on infected plants from irdefensin line 76 (p = 0.012) and irdefensin line 96 (p = 0.045). The larvae that fed on PST DC3000-infected irdefensin (76 and 96) plants were smaller than the larvae that fed on PST DC3000-infected WT plants (Fig. 3B).
The large variation in the larval mass across the experiment could be
attributed to the differences in larvae's development which in turn may
be attributed to high spatial heterogeneity in food quality for the
larvae consuming infected leaves.
Figure 3. Pst DC3000 inoculation and Nadefensin silencing decrease M. sexta larval mass gain in N. attenuata. A). Mean (± SE) M. sexta larval mass gain on WT plants and irdefensin lines
76 and 96. A neonate larva was placed in a clip cage and allowed to
feed continuously for 12 days. Larval mass was recorded on days 6, 9
and 12. B) Photographs taken after 12 days of M. sexta feeding on WT and irdefensin lines 76 and 96 that were induced with Pst DC3000. Asterisk indicates significant differences (p = 0.05) between WT and irdefensin lines (76 and 96) after Pst DC3000 infection (N = 30).
Detecting PST DC3000 from infected plants in herbivores' guts
In our earlier work, we reported that irdefensin (76 and 96) plants were more susceptible to PST DC3000 than were WT N. attenuata plants, and as a result irdefensin (76 and 96) plants contained more PST DC3000 colony forming units (CFUs) than did the WT plants . In this study we observed that M. sexta larvae that fed on irdefensin (76 and 96) plants were smaller and seemed to be infected with pathogens (Fig. 3B). Therefore, we hypothesized that herbivores feeding on PST DC3000-infected irdefensin (76
and 96) plants might have ingested more PST DC3000 than did the larvae
feeding on the PST DC3000-infected WT plants, and that the number of
ingested PST DC3000 might negatively correlate with larval growth. We
counted the CFUs of plant-derived PST DC3000 in guts (including the
foregut, midgut and hindgut) of larvae that fed either on PST
DC3000-infected WT plants or PST DC3000-infected irdefensin plants (76 and 96) (Fig. 4A and 4B). As expected, we found PST DC3000 colonies in larvae that fed on PST DC3000-infected WT and irdefensin (76 and 96) but none in larvae that fed on uninfected WT and irdefensin (76 and 96) plants. However, the number of PST DC3000 colonies in the guts of larvae that fed on PST DC3000-infected WT or irdefensin (76 and 96) plants did not differ significantly (Fig. 4A; ANOVA, F5,24 = 2.07, P =
0.104). Moreover, the overall number of CFUs was very low relative to
the number of CFUs found in leaves, which suggests that plants infected
with PST DC3000 do not detrimentally affect larvae by directly
transmitting pathogens to the herbivores. In addition to PST DC3000, we
also detected a few unknown microorganisms with resistance to
tetracycline and rifamycin (the selection markers for PST DC3000).
Interestingly these unknown microorganisms were found most often in
guts extracted from larvae that fed on irdefensin (76 and 96) plants. irdefensin (76
and 96) plants also show an increased susceptibility to the many
opportunistic microorganisms which may be detrimental to larvae as well
Figure 4. The number of Pst DC3000 colonies quantified in the guts of M. sexta larvae that fed on the Pst DC3000-inoculated WT and irdefensin (76 and 96) plants do not differ. A) Mean (± SE) colony-forming units (CFUs) of Pst DC3000 in the guts of larvae that fed on either the Pst DC3000-inoculated WT/irdefensin (76 and 96) or uninduced WT/irdefensin (76 and 96) plants. The larval guts from 5 replicate larvae that fed on either Pst DC3000-inoculated WT and irdefensin (76 and 96) for 12 days were surgically removed and ground in 1 ml sterile water. 40 μl
of supernatant was spread on plates containing LB agar plate containing
rifamycin and tetracycline to select for the growth of Pst DC3000.
Colonies were counted after 48 h of incubation at 28°C. B) Photographs
of LB plates + antibiotics (rifamycin and tetracycline) showing Pst
DC3000, in addition to four unidentified/unknown microorganisms that
could also grow on LB plants supplemented with antibiotics (N = 5).
Effects of herbivory and Nadefensin silencing on PST DC3000 infection
Silencing Nadefensin expression in N. attenuata does not influence the plant's resistance to M. sexta attack but lowers resistance to PST DC3000 , which suggests that Nadefensin functions as an antibacterial defense protein in N. attenuata. We therefore explored whether Nadefensin still
functions as an antibacterial protein in leaves that are damaged by
herbivores. We compared the level of disease progression of PST DC3000
in leaves that were either undamaged or previously damaged (4 days) by M. sexta.
Two and four days after PST DC3000 infection, leaves were evaluated for
CFUs. In general, we found that inoculating leaves of undamaged plants
with PST DC3000 or infecting leaves of M. sexta-damaged (4
days of feeding) plants with PST DC3000 resulted in statistically
significant differences in PST DC3000 growth responses in N. attenuata (Fig. 5; ANOVA, F17,72 = 128.75, P <
0.001). Investigating the genotypic and treatment effects, we found the
following patterns on day 4: 1) PST DC3000 CFUs were higher in both
uninduced irdefensin line 76 (9%; p = 0.031) and line 96 (6.6%; p = 0.047) than in uninduced WT plants; 2) similarly, PST DC3000 CFUs were higher in M. sexta-damaged plants from both irdefensin lines 76 (11.3%; p = 0.014) and line 96 (8.9%; p = 0.048) than in WT plants; 3) within the WT plants, control plants (undamaged) had higher titers of PST DC3000 CFUs (10.6%; p = 0.017) than did M. sexta-damaged plants; 4) within the irdefensin lines (76 and 96) the same effects of Manduca damage were observed: control plants (undamaged) had a higher titers of PST DC3000 CFUs, 8.53% (p = 0.037) and 8.51% (p = 0.0183), respectively, than did the M. sexta-damaged lines; 5) control WT plants (T4-undamaged) and M. sexta-damaged irdefensin lines 76 and 96 did not differ in PST DC3000 CFUs (p = 0.934 and p =
0.676, respectively). In summary, these results suggest that
Na-defensin's anti-bacterial defense property is retained in leaves
regardless of whether a leaf is elicited by pathogen or herbivore. In
addition, M. sexta damage which results in the elicitation of
a large set of anti-herbivory defense metabolites more effectively
restricted PST DC3000 growth than did elicitation by Na-defensin alone.
Figure 5. M. sexta feeding significantly reduces Pst DC 3000 disease spread. Values are mean (± SE) colony-forming units (CFUs) of Pst DC3000 after inoculation of the leaves of WT/irdefensin (76 and 96) plants that were either uninduced or previously attacked by M. sexta larvae 4 days earlier. To record the CFUs, surface-sterilized leaf discs (1 cm2) were ground in 1 ml sterile water and 40 μl
of supernatant was spread on plates containing LB agar + antibiotics
(rifamycin and tetracycline). Colonies were counted after 48 h of
incubation at 28°C. Different letters in lower and upper cases indicate
significant differences among Pst DC3000-inoculated WT plants and the
transgenic plants on days 2 and day 4, respectively (N = 5).