EVALUATION OF A BIOTECHNOLOGICAL TOOL FOR THE CONTROL OF
PECTOBACTERIUM CAROTOVORUM, CAUSING AGENT OF SOFT ROT
Fullana, Lara; Krapp, Adriana R.; Carrillo, Néstor; Arce, Rocío C.
Instituto de Biología Molecular y Celular de Rosario (IBR-UNR/CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario
ABSTRACT
Iron incorporation is problematic for terrestrial organisms, and many infectious agents have developed mechanisms to hack host ferroproteins. The most abundant iron-containing
plant protein is ferredoxin (Fd), which delivers redox equivalents to numerous essential oxido-reductive pathways, including photosynthesis. One of the phytopathogens that have
taken advantage of this abundance to their benefit is Pectobacterium carotovorum (Pcc), a necrotrophic bacterium that causes soft rot in numerous crops of agro-economic
importance, such as potatoes and onions. Several observations showed that this mechanism of iron acquisition via Fd could be key to the success of Pcc infection, suggesting that
plants with decreased Fd levels could be resistant to soft rot. Unfortunately, Fd is an essential gene, and its deficiency has catastrophic phenotypic consequences for the plant. In this
context, many cyanobacteria and algae contain an iron-free protein called flavodoxin (Fld), which can functionally replace Fd, and whose expression is induced in situations of iron
starvation. Although the Fld-encoding gene is absent from the plant genome, the introduction of a cyanobacterial Fld directed to chloroplasts increased tolerance to environmental
stresses including iron limitation. Therefore, we tested the virulence of Pcc in transgenic plants with decreased levels of Fd and expressing Fld. We worked on leaves of two plant
species in which Fd levels were decreased by different methods: Arabidopsis knockout mutants in the gene encoding the major leaf ferredoxin (Fd2); and tobacco plants using
transient virus-induced gene silencing (VIGS). We evaluated the disease progression by monitoring the onset of symptoms and their spread, estimation of bacterial populations and
electrolyte leakage in leaf tissue. Our results show that Fd deficiency would limit the availability of iron during Pcc infection and affect the development of the disease.
RESULTS
Arabidopsis thaliana
First, the levels of Fd in both, wild-type (WT) and Fd2-KO Arabidopsis lines were determined (Figure 1). Quantitative analysis revealed that the mutant plants contain a halving of the
protein dose of Fd compared to the WT. Subsequently, transgenic Arabidopsis plants expressing Fld directed to chloroplasts were generated in WT and Fd2-KO backgrounds. Plants
were transformed with both the vector pCAMBIA3200_pfld and with the empty vector, pCAMBIA3200 as a control, generating 4 new lines:
• : Fd2-KO plant with thNos-pCAM: WT with empty vector pCAMBIA3200 • Nos-pfld: WT with pCAMBIA3200_pfld vector
• Fd2-KO-pCAMe empty vector pCAMBIA3200 • Fd2KO-pfld: Fd2-KO plant with pCAMBIA3200_pfld vector
These lines were characterized following some physiological parameters. Plants with Fd2 knocked out presented differential characteristics regarding to wild-type lines, such as lower
production of leaves and a smaller rosette size (Figure 2). These results are consistent with what is reported in the literature (Voss et al. 2008, Physiol. Plant. 133: 584-598). The
presence of Fld ameliorated the negative effects due to absence of Fd in terms of phenotype (Figure 2).
Leaves of the different lines generated were infected with Pcc in order to analyze whether the decrease in Fd is capable of limiting the infection potential of the pathogen. The
progression of the infection was monitored over time, where although the symptoms began to develop at 15 hours post infection (hpi), no statistically significant differences were
observed between the lines at that time (Figure 3). On the other hand, at 18 hpi, the lines that have knocked out the Fd2 gene, regardless of Fld expression, were the ones that best
maintained the integrity of the leaf, and therefore, the ones that suffered the least damage when faced with Pcc (Figure 3).
Nos-pCAM
Relative intensity
Relative integrity
Nos-pfld
WT
Fd2KO-pCAM
Fd2KO-pfld
WT
Figure 1. Characterization of Fd2-KO lines. Western blot Figure 2. Images of the 4 lines used for the Figure 3. Progression of infection with Pcc in leaf and quantification relative to a pre-
with antibodies against Fd and quantification by ImageJ. analysis over time. symptomatic time of the preserved area over time.
Nicotiana tabacum - VIGS
The VIGS approach to gene silencing in tobacco plants was developed by knock downing the phytoene desaturase gene that causes leaf whitening that can be seen with the naked
eye (Caplan and Dinesh-Kumar 2006, Curr. Protoc. Microbiol. 16: 16I.6). We tested different infiltration protocols (Figure 4):
- Syringe infiltration. Leaves are infiltrated from the abaxial part with blunt syringes containing the infiltration solution.
- Agrodrench. In this method, the infiltration solution is poured into the crown of the plant.
The technique that had the highest efficiency was syringe infiltration, so it was decided to follow the subsequent infiltrations following this methodology.
Next, Fd silencing was performed by VIGS using wild-type plants and plants with different levels of this protein were obtained. It was observed a correlation between the decrease in
Fd and the lower progression of the disease by the pathogenic bacteria (Figure 5).
Syringe Agrodrench
1 2 3
Figure 4. Silencing of the phytoene desaturase gene in tobacco plants, as a positive Figure 5. Photographs of Pcc-infected wild tobacco plant leaves at 48 hpi and Western blot of
control for VIGS technique. protein extracts from tobacco leaf discs revealed with an antibody against Fd.
CONCLUSIONS
▪ Arabidopsis lines with decreased levels of Fd2 exhibited less foliar damage against Pcc infection.
▪ Lower Fd contents in tobacco plants silenced by VIGS method correlated with a lower progression of the infection.
Our results show that the deficiency in Fd would limit the availability of Fe during infection with Pcc and affect the development of the disease.
