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First published online June 3, 2009; 10.1104/pp.109.141523 Plant Physiology 150:1713-1722 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Dissection of Bacterial Wilt on Medicago truncatula Revealed Two Type III Secretion System Effectors Acting on Root Infection Process and Disease Development[C],[W],[OA]Université de Toulouse, INPT, Laboratoire Symbiose et Pathologie des Plantes, ENSAT, BP 31607 Auzeville-Tolosane, 31326 Castanet-Tolosan, France (M.T., M.-J.T., F.V., L.G., M.-F.J.); Surfaces Cellulaires et Signalisation chez les Végétaux, UPS-CNRS UMR 5546, 31326 Castanet-Tolosan, France (A.J.); and Laboratoire des Interactions Plantes Microorganismes, INRA/CNRS UMR 441/2594, 31326 Castanet-Tolosan, France (S.G.)
Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease, which colonizes susceptible Medicago truncatula via the intact root tip. Infection involves four steps: appearance of root tip symptoms, root tip cortical cell invasion, vessel colonization, and foliar wilting. We examined this pathosystem by in vitro inoculation of intact roots of susceptible or resistant M. truncatula with the pathogenic strain GMI1000. The infection process was type III secretion system dependent and required two type III effectors, Gala7 and AvrA, which were shown to be involved at different stages of infection. Both effectors were involved in development of root tip symptoms, and Gala7 was the main determinant for bacterial invasion of cortical cells. Vessel invasion depended on the host genetic background and was never observed in the resistant line. The invasion of the root tip vasculature in the susceptible line caused foliar wilting. The avrA mutant showed reduced aggressiveness in all steps of the infection process, suggesting a global role in R. solanacearum pathogenicity. The roles of these two effectors in subsequent stages were studied using an assay that bypassed the penetration step; with this assay, the avrA mutant showed no effect compared with the GMI1000 strain, indicating that AvrA is important in early stages of infection. However, later disease symptoms were reduced in the gala7 mutant, indicating a key role in later stages of infection.
Very few phytopathogenic bacteria colonize intact plant roots, even if the local soil provides a favorable environment for bacterial growth. Penetration seems to be the limiting step, as most bacteria need natural openings or wounds to invade their hosts and there are few opportunities in roots (Loria et al., 2003  ). Ralstonia solanacearum, a gram-negative soil-borne pathogen responsible for bacterial wilt, naturally invades plants through axils of secondary roots. R. solanacearum has to attach to the root, find nutrients, multiply, migrate into plant tissues, and penetrate the xylem in order to cause disease (Vasse, 1995). It has a wide and still-expanding host range that includes several hundred susceptible species in at least 50 different plant families, which include economically important hosts such as potato (Solanum tuberosum), tomato (Solanum lycopersicum), peanut (Arachis hypogaea), banana (Musa spp.), pepper (Capsicum annuum), eggplant (Solanum melongena), and tobacco (Nicotiana tabacum; Hayward, 1991). The high incidence of plant mortality and the lack of effective control methods make R. solanacearum one of the world's most destructive plant pathogens (Prior et al., 1998). This bacterium is a model organism extensively studied by genetic, molecular, and biochemical techniques in order to develop strategies for the control of bacterial wilt.
R. solanacearum virulence responds to several environmental stimuli and involves the production of multiple virulence factors (Schell, 2000
Like many other pathogenic bacteria, R. solanacearum possesses a type III secretion system (T3SS), encoded by the hypersensitive response and pathogenicity (hrp) genes. In contrast to T2SS, which secretes degrading enzymes, the T3SS allows the injection of effector proteins directly into the plant cell through the hrp pili. The T3SS is essential for pathogenicity, since R. solanacearum hrp mutants cannot induce disease in many susceptible plants such as tobacco, tomato, petunia (Petunia hybrida), and Medicago (Vasse et al., 2000
Some T3Es are virulence factors: they promote pathogenic functions of the bacteria and disrupt defense responses of the host immune system. Several mechanisms by which T3Es suppress plant defense have been described, including the inhibition of plant signaling components and the alteration of host transcription or posttranscriptional processes. Some T3Es are proteases that can degrade defense components through the host ubiquitination machinery and the 26S proteasome (Block et al., 2008
This work aims to study the sequence of events related to the T3SS-dependent infection process of R. solanacearum on intact plant roots. Many previous studies of plant-R. solanacearum interaction involved bacteria delivered directly into the vascular system through artificially wounded roots, in order to increase penetration potential (Deslandes et al., 1998
The Interaction between R. solanacearum and M. truncatula Leads to T3SS-Dependent Root Symptoms
In order to study the infection process of R. solanacearum in M. truncatula, we used an in vitro inoculation procedure of intact roots adapted from Vailleau et al. (2007) Viability of the epidermis and root hairs was monitored by fluorescein diacetate (FDA), which stains living cells (Fig. 1, A, D, and G). The epidermis of the mock inoculation control was viable, as evidenced by green fluorescence. Also, root hair initiation and elongation occurred (Fig. 1, A–C). After inoculation with GMI1000, a loss of fluorescence was first observed at 24 hpi and was complete at 72 hpi, indicating a loss of viability of the root tip epidermis (the root tip was considered the first 2–3 mm of the root; Fig. 1G). In addition, no root hair initiation or elongation was detected, and root tip browning and swelling were observed (Fig. 1, H and I). Root growth arrest, root tip swelling, browning, and epidermis loss of viability will be subsequently referred to "root symptoms."
Despite the similarity in the root symptoms between resistant and susceptible lines, foliar symptoms were only observed in the susceptible line (A17). The chlorosis and wilting of leaves and cotyledons led to the death of an average of 50% of A17 plants when the experiment was terminated 28 d post inoculation (dpi). Infection with R. solanacearum strains carrying mutations in the regulatory hrpB gene (GMI1525) or in the structural T3SS gene hrcS (GMI1402) resulted in the absence of root growth arrest at 24 hpi, an intact root tip epidermis at 72 hpi, no swelling and browning at 7 dpi (Fig. 1, D–F), and no wilting of the shoots up to 28 dpi. Root and foliar symptoms, therefore, were dependent on a functional T3SS. Bacterial mutants defective in EPS or T2SS biosynthesis were altered in their ability to induce foliar symptoms but induced root symptoms similar to those caused by GMI1000 (data not shown). Therefore, root symptoms were specifically dependent on T3SS.
The bacterial distribution and the internal structure of the root tip were observed at 3 and 7 dpi on the susceptible line using semithin resin sections (Fig. 2).
After mock inoculation, the root tip was typically organized (Fig. 2A; Groot et al., 2004
In order to quantify the differential bacteria colonization, infections were conducted using a R. solanacearum strain transformed with a constitutive GUS construct (GMI1559; Etchebar et al., 1998 ). The GUS activity was revealed in planta, and the observations were performed with a stereomicroscope. At 14 dpi, bacteria had crossed the epidermis and colonized the cortical root tip cells of 45% of the F83005.5 plants and 85% of the A17 plants. Bacteria were never observed in the vessels of F83005.5, whereas 55% of the A17 plants had their vessels colonized at 14 dpi. Thus, two stages in the infection process were clearly identified. The first one, common to the susceptible and the resistant lines, was the bacterial colonization of the root tip cortical cells. Interestingly, a restriction of bacterial propagation was found in the resistant line F83005.5, indicating the possible onset of resistance. The second stage, specific to the susceptible line, was the colonization of the vascular bundles, leading to plant death.
To identify potential cell wall modifications in response to infection, cytological studies were performed on transverse root tip sections of the susceptible and resistant lines after pathogen infection. Autofluorescence of the inner root tip and callose, suberin, and lignin deposits were observed 3 and 7 dpi in both A17 and F83005.5 lines after GMI1000 (Fig. 3) or mock (data not shown) inoculation. Autofluorescence of the cell wall of the cortical cells was observed only after inoculation (Fig. 3). Sections with (Fig. 3A) or without (Fig. 3B) cell wall autofluorescence were observed in both resistant and susceptible roots; however, at 7 dpi, roots of the resistant line showed cell wall autofluorescence at a significantly higher proportion than was observed in the susceptible line (frequency test, P = 0.027).
Callose and suberin accumulations were observed equally in susceptible and resistant lines (data not shown). Lignin deposits were observed in the endodermis of the two lines only after inoculation with the GMI1000 strain (Fig. 3). Sections with (Fig. 3C) or without (Fig. 3D) endodermis lignifications were observed in the inoculated roots, but at 3 dpi, F83005.5 roots showed lignin deposits on the endodermis, at a significantly higher frequency (frequency test, P = 0.011) than did A17 roots. Interestingly, at 7 dpi, A17 and F83005.5 root tip endodermis were lignified to a similar extent (frequency test, P = 0.366), indicating that rapid lignification may be important in resistance.
As seen previously, root symptoms were specifically dependent on the presence of the T3SS. To identify bacterial molecules involved in the development of these root symptoms in the susceptible line, we used FDA staining to screen a collection of 67 loss-of-function T3E mutants in the GMI1000 background (Supplemental Fig. S1; Poueymiro et al., 2009
In order to uncover a potential additive effect of the two effectors on the root symptom, an avrA and gala7 double mutant strain was generated. The FDA test revealed a median score of 2, showing an additive effect on epidermal cell death (Wilcoxon rank sum test, P = 2.10–4; Fig. 4). Root growth measurements showed high variability, and no additive effect could be demonstrated.
Among the effector mutants showing a minor effect (Supplemental Fig. S1), two of them were selected: GRS118 (GMI1000
After observing the involvement of AvrA and Gala7 in root symptom development after R. solanacearum inoculation, we characterized their roles in root bacterial colonization and the development of foliar symptoms. At 14 dpi, the gala7 mutant was never detected in the root tip cortex and therefore did not colonize the vascular system (Fig. 5), nor did it induce wilting of the A17 plant (Fig. 6). This indicated that Gala7 was necessary for the bacterial penetration into the root of the susceptible M. truncatula line.
At 14 dpi, the avrA mutant strain was found in the root tip cortex of 18% of the plants and in the vascular system of 4% of the plants, in comparison with GMI1000, which was found in the root tip cortex of 85% of inoculated plants and in the vascular system of 55% (Fig. 5). The plant wilting rate was also 5 times lower following inoculation with avrA compared with GMI1000 (Fig. 6). At 28 dpi, unwilted plants did not show any sign of infection. Therefore, the avrA mutant was not delayed in the induction of wilting symptoms but showed a reduced aggressiveness in comparison with GMI1000 strain. All wilted plants inoculated with the avrA mutant showed root growth arrest similar to that found after GMI1000 inoculation. Therefore, AvrA appears to be not essential for the root penetration of the susceptible M. truncatula line, but it does play a positive role in this process. In conclusion, the effectors Gala7 and AvrA are involved in the appearance of root symptoms and also play roles in root colonization and disease development. Gala7 was necessary for bacterial penetration of root cells, whereas AvrA was partially required for root colonization.
After showing the involvement of the two effectors in root penetration, it was interesting to assay their role in the subsequent steps of vascular propagation and wilting development. Therefore, we used an inoculation procedure that bypassed root penetration using root tips that were cut just before the bacterial inoculation (Vailleau et al., 2007
In order to study the root infection process of the devastating pathogen R. solanacearum, we developed an in vitro inoculation procedure using intact M. truncatula roots. The root tip was identified as the key site for T3SS-dependent root symptom development and root penetration of R. solanacearum. The pathosystem consisted of susceptible and resistant M. truncatula lines infected with the R. solanacearum strain GMI1000. Examination of this pathosystem allowed the dissection of sequential stages of pathogen infection and the identification of two effectors important in this process.
The first step in the infection (Fig. 8)
was the development of root symptoms, which included root growth arrest, root tip swelling and browning, and epidermal cell death. These root symptoms were not dependent on the plant genetic background, as they were common to both susceptible and resistant lines. They were also observed on other M. truncatula lines infected with R. solanacearum, four resistant (TN7.22, TN3.17, TN6.17, and DZA315.16) and three susceptible (TN1.21, TN9.1, and TN6.2) lines. They appeared to be the first step in bacterial infection, but they were not always associated with the wilting of the plant. The progression of early steps of the infection processes is not always associated with resistance, as described in other systems (Dita et al., 2007
R. solanacearum infection processes in intact roots have been described in tomato (Vasse, 1995 ) and petunia (Zolobowska and Van Gijsegem, 2006). No tomato root symptoms were mentioned. However, in petunia, Zolobowska and Van Gijsegem (2006) observed swelling of the root tip, reduced elongation of main and lateral roots, inhibition of lateral root development, and formation of root lateral structures. Similarly, we found M. truncatula root growth arrest, which could result from the disruption of the root meristem structure that was observed. At the same time, internal cell maturation may still occur, as illustrated by the enlarged vacuolated cells and supernumerous mature vessels normally encountered farther from the root tip (Fig. 2B). However, root alterations may not be a specific virulence mechanism but may just be a consequence of infection.
Root tip epidermal cell death, caused by effectors secreted by the bacteria, could be beneficial to R. solanacearum. It could lead to the release of specific attractant compounds, as chemotaxis has been described to play a role in fitness and virulence (Yao and Allen, 2006
The specific requirement of a functional T3SS for root symptom development led us to test a panel of T3E mutants for their ability to cause epidermal cell death. This screen resulted in the identification of two effectors: Gala7 and AvrA. These two effectors were involved in epidermal cell death and had an additive effect. Other effectors may be involved to a lesser extent, as the root tip epidermis was still partially altered after infection with the double mutant strain. The two effectors were also involved in root growth arrest, but no significant additive effect could be measured due to the variability of root growth. These effectors were both demonstrated to be translocated into plant cells by the T3SS (Angot et al., 2006
The second step in the infection process (Fig. 8) was the bacterial invasion of root tip cortical cells. The root tip was the only zone where we found that the bacteria could penetrate into M. truncatula through intact root tissue and not through induced or natural wounds, like axils of secondary roots on tomato (Vasse, 1995
Cortical cell invasions occurred in both M. truncatula lines but were reduced in the resistant line. The cell wall is the primary interface for pathogen interactions and therefore is an important site of potential defense mechanisms, such as cell wall deposition and strengthening (Cano-Delgado et al., 2003 At this stage, the bacterial effectors were the major determinants of infection, as the gala7 mutant was never detected in cortical cells of line A17 and avrA mutant presence was strongly reduced. Our inoculation procedure highlights a new role for Gala7 as a determinant of bacterial penetration.
The third step in the infection process (Fig. 8) was bacterial propagation in the vascular tissue. The avrA mutant was detected in vessels of A17 plants, but at a much lower rate than GMI1000, showing bacterial determination at that stage. Plant genetic background was also important at that stage, as GMI1000 was never detected in the vascular tissue of F83005.5 plants. As seen previously, we detected autofluorescent cell wall cortical cells, suggesting the presence of phenolic compounds. Therefore, we assayed for the presence of lignin, a phenylpropanoid known to strengthen the cell wall and inhibit further pathogen ingress (Hijwegen, 1963
This third stage was tightly linked to the next step (wilting development; Fig. 8). No wilting was observed when bacteria did not reach the vessels. This suggests that foliar symptom development depends on the bacterium's ability to penetrate the root tip vasculature in M. truncatula, which was not described before in other systems. Indeed, bacteria could be detected in the vascular system of resistant tomato plants, but at a low level that does not induce plant wilting (Grimault et al., 1995
The avrA mutant strain was able to reach the same stages as GMI1000, but to a lesser extent at each step; therefore, it had a reduced pathogenicity on M. truncatula. Although the AvrA gene is widely distributed in R. solanacearum, disruption of avrA in GMI1000 strain did not lead to a reduced pathogenicity on susceptible hosts such as tomato and Arabidopsis (Poueymiro et al., 2009
The gala7 mutant never colonized intact roots and therefore never induced foliar wilting, whereas the avrA mutant showed reduced aggressiveness. Therefore, we wondered if those two effectors were also involved in the virulence of R. solanacearum after xylem invasion and wilting symptom development. Invasion of the bacteria was made possible by cutting the root tip at the time of inoculation. In this condition, the avrA mutation did not affect disease development. In contrast, the gala7 mutation led to a reduction of wilting symptoms. These results suggested that the two effectors have different roles in R. solanacearum virulence, with AvrA only involved in the penetration steps.
The effects of the two T3Es have been studied on both root symptom development and disease development of leaves of the susceptible line, and two different mechanisms were revealed. The involvement of the two T3Es differed between early and later stages in infection: they had additive effects on root tip epidermal cell death, whereas disease development in vascular tissues involved gala7 but not avrA. Root penetration likely requires a large number of molecules, consistent with the fact that the development of root symptoms involves a nonspecific mechanism, observed on both resistant and susceptible lines and, at least partially, on petunia (Zolobowska and Van Gijsegem, 2006
Bacterial Strains and Culture Conditions
The wild-type strain GMI1000 of Ralstonia solanacearum and hrp mutant derivatives (GMI1525, a hrpB regulatory mutant, and GMI1402, a hrcS T3SS structural mutant) were described previously (Cunnac et al., 2004
An avrA deletion mutant strain was generated by inserting the
Seeds were surface sterilized, vernalized, and germinated according to Boisson-Dernier et al. (2001)
Inoculation was performed as described previously (Vailleau et al., 2007
Roots were measured every 24 h up to 15 dpi with 10 plants per treatment. Root tip browning and deformation were observed using a stereomicroscope (Leica MZ75 equipped with a CCD camera [Leica DCF320]).
GUS staining was performed on 10 plants per sample as described by Jefferson (1987)
The FDA viability test was performed as described previously (Heslop-Harrison and Heslop-Harrison, 1970 For semithin sections, fresh material was fixed in 0.1 M sodium cacodylate and 2.5% glutaraldehyde, dehydrated twice for 1 h each time in 20%, 40%, 60%, 75%, 80%, 90%, and 100% ethanol, progressively infiltrated in medium-grade LR white resin (48 h in 1:3 LR white resin and 2:3 ethanol, 48 h in 1:2 LR white resin and 1:2 ethanol, 48 h in 2:3 LR white resin and 1:3 ethanol, and 48 h in pure LR white resin), and encapsulated for resin polymerization for 12 h at 65°C. Sections of 1 µm were made with Ultracut E (Reichert-Jung) and stained during 30 s at 65°C with 0.1% toluidine blue in 2.5% Na2CO3 at pH 11. Three roots were sectioned per biological replicate.
Root tip swelling and cell wall modifications were assayed on fresh material included in 5% low-melting agar (agarose type I; Sigma) and sectioned with a vibratom (Leica VT 1000S). Phloroglucinol (hydrochloric solution; VWR Prolabo) coloration (10 min) was used to detect lignin (violet) and suberin (pink) deposits; callose detection was performed using 0.1% aniline blue in 0.15% K3PO3, pH 12.4 (10 min), adapted from Pérez-de-Luque et al. (2007) All images were obtained using an inverted microscope (Leica DMIRBE), and images were acquired with a CCD camera (color cooled view; Photonic Science). For the FDA test, fluorescence was obtained using excitation range 450 to 490 nm, dichroic mirror 510 nm, and long-pass emission filter 515 nm. For the autofluorescence test and callose detection, excitation range 340 to 380 nm, dichroic mirror 400 nm, and long-pass emission filter 425 nm were used.
The following materials are available in the online version of this article.
We thank Jean Greenberg, Jennifer Mach, and Marc Ellis for reading and comments on the manuscript, Jean-Marie Prospéri for providing A17 and F83005.5 seeds, and the IFR40 RIO platform for microscopy experiments. Received May 15, 2009; accepted May 28, 2009; published June 3, 2009.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Marie-Françoise Jardinaud (jardinaud{at}ensat.fr).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.141523 * Corresponding author; e-mail jardinaud{at}ensat.fr.
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ABSTRACT
RESULTS
RSp0846-RSp0847) and GRS230 (GMI1000 
interposon carrying a spectinomycin resistance gene (Prentki and Krisch, 1984