Effect of Phytophthora infestans in the physiology and induction of pathogen related proteins in Physalys peruviana

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(1)1 . Effect of Phytophthora infestans in the physiology and induction of pathogen related. 2 . proteins in Physalys peruviana. 3 . Antolinez, A., Danies, G., Cantillo, J., Peña, G., Vargas, A.M., Bernal., A. and Restrepo. 4 . S*.. 5 . Laboratorio de Micología y Fitopatología, Universidad de los Andes, Bogotá, Colombia. 6 . * Corresponding author. Mailing address: Laboratorio de Micología y Fitopatología. 7 . (LAMFU) J205, Universidad de los Andes, Cra. 1 N° 18A - 10 Bogotá–Colombia.. 8 . Phone: +(571) 3394949 Ext: 3772. E-mail: [email protected]. 9 10 . 1 .

(2) 11 . Abstract. 12 . Phytophthora infestans is a plant pathogen that affects a great variety of crops within the. 13 . Solanaceae family. In 2007 the pathogen was described causing disease in Physalis. 14 . peruviana (cape gooseberry). Since the report of the disease, we have studied this particular. 15 . interaction. The aim of this work was to characterize the first defense reactions produced on. 16 . cape gooseberry leaves due to the infection with P. infestans. Detached cape gooseberry. 17 . leaves were inoculated with a solution of 103 sporangia/ml. Electron microscopy. 18 . photographs were taken at 0, 24, 48, 72 and 96 hours post inoculation on the leaf, abaxial. 19 . surface up. Reactive oxygen species (ROS), and the induction of Pathogenesis Related (PR). 20 . proteins were measured after inoculation. We also performed a qRT-PCR in order to. 21 . investigate the molecular bases of this interaction. We have evidenced sporangial. 22 . germination and active aerial growing of Phytophthora on the leaf surface. There is an. 23 . induction in the expression of carbonic anhydrase and glucanase A genes, as well as other. 24 . markers related to the plant defense response. In particular, we detected the induction of the. 25 . enzymes peroxidase and glucanase as well as the production of ROS. To our knowledge,. 26 . this is the first study that characterizes the first biochemical reactions caused by P. infestans. 27 . on cape gooseberry. Our results are fundamental for understanding how P. infestans affects. 28 . other members of the Solanaceae family.. 29 . Keywords. 30 . Phytophthora infestans, Physalis peruviana, plant defense, physiology.. 2 .

(3) 31 . Introduction Phytophthora infestans is the causal agent of late blight, a plant disease that causes. 32 33 . multibillion dollar loses in potato and tomato crops worldwide. (Vleeshouwers, van. 34 . Dooijeweert et al. 2000; Fry 2007; Runno-Paurson, Fry et al. 2010). Interestingly the. 35 . pathogen is also able to attack a wide range of plants within the Solanaceae family such as. 36 . Nicotiana benthamiana, and Andean exotic fruits such as Solanum betaceum and Solanum. 37 . quitoense (Vargas, Correa et al. 2009). The degree of susceptibility varies among species. 38 . and even among varieties in the same species (Vleeshouwers, van Dooijeweert et al. 2000;. 39 . Smart, Myers et al. 2003). In spite of the extensive research on this interaction, until now,. 40 . some of the plant defense mechanisms are not completely understood. However it is well. 41 . known that a variety of mechanisms such as cell wall reinforcements (Vleeshouwers, van. 42 . Dooijeweert et al. 2000), phytoalexin production (Hammerschmidt 1999), expression of. 43 . pathogen-related proteins and generation of reactive oxygen species (ROS) are part of the. 44 . defense response (Apel and Hirt 2004). The hypersensitive reaction (HR) a type of. 45 . programmed cell death, also plays an important role on disease resistance avoiding the. 46 . development of the pathogen (Scharte, Schon et al. 2005).. 47 . In 2007 Vargas et al, reported P. infestans causing disease in a new host, Physalis. 48 . periviana (cape gooseberry), an Andean exotic fruit that in the last decade has become of. 49 . great importance in the Colombian international export market (Novoa, Bojacá et al. 2006).. 50 . Interestingly since the report of the disease the pathogen has not caused significant. 51 . economically loses for cape gooseberry growers and the symptoms of the disease have been. 52 . seen sporadically in the field. Even more interesting is the fact that cape gooseberries are. 53 . usually grown in the same fields as potato, where the pathogen is constantly present 3 .

(4) 54 . causing severe epidemics (Vargas, Correa et al. 2009). The mechanisms that make these. 55 . interactions so different remain unclear and no information about cape gooseberry. 56 . responses or alterations in its physiology due to P. infestans have been documented.. 57 . Here, we examined a broad range of physiological responses in order to understand whether. 58 . the differences in susceptibility between cape gooseberry and potato were the result of. 59 . defense mechanisms activated in cape gooseberry in order to avoid tissue infection. We. 60 . hypothesized that the lack of symptoms was caused by an activation of a rapid defense. 61 . response triggering ROS production and PR induction. Consequently we performed an in. 62 . situ staining of ROS and enzymatic activity assays of three well known PR proteins. We. 63 . also performed a qRT-PCR in order to identify the induction of the salicylic acid pathway. 64 . and the induction of genes involved in the antioxidative system. These tests will provide. 65 . evidence to understand how other solanaceous hosts are responding to P. infestans.. 66 67 . Materials and methods. 68 . Plant material, pathogen isolates and inoculation.. 69 . Four week-old cape gooseberry plants (Physalis peruviana) were grown under. 70 . greenhouse conditions in plastic bags with a soil supplemented with fertilizer containing. 71 . nitrogen, phosphorus and potassium (8-8-8) and irrigated every two days. Plants were. 72 . randomly chosen and sorted into two groups with 30 plants each. Two days prior to the. 73 . inoculation plants were transplanted to an infection chamber under natural sunlight in the. 74 . greenhouse with a photoperiod of 12h approximately. One group of plants was inoculated. 75 . with Phytophthora infestans isolates 21, Z3-2 (A1 mating type isolated from Solanum 4 .

(5) 76 . tuberosum), and 2557 (A1 mating type isolated from Physalis peruviana) belonging to the. 77 . clonal lineage EC-1 from the collection of the Laboratory of Mycology and Plant. 78 . Pathology, Universidad de los Andes, Bogotá, Colombia grown in rye agar and the second. 79 . group was inoculated with tap water (mock inoculated control). P. infestans sporangia for. 80 . inoculations were obtained by washing two-week old cultures grown on rye B agar with. 81 . sterile distilled water. The final concentration was determined using a hemocytometer and. 82 . the suspension was then chilled at 4oC for 2 h to induce zoospore release. Plants were. 83 . sprayed with a suspension of 30.000 of P. infestans sporangia per ml, until run-off (Smart,. 84 . Willmann et al. 1998). Leaves from inoculated and mock treatments were sampled at. 85 . different times post inoculation in order to perform the different assays.. 86 87 . Histochemical detection of H2O2.. 88 . The detection of the accumulation of hydrogen peroxide in cape gooseberry leaves. 89 . was performed using an in situ staining using 3,3 Diaminobenzidine (DAB) as a substrate. 90 . (Sigma-Aldrich Inc., St. Louis, MO), according to the methods described by (Orozco-. 91 . Cardenas and Ryan 1999), with some modifications. Detached cape gooseberry leaflets. 92 . from inoculated and mock-inoculated plants at 0, 12, 24, 48, 72 and 96 hours post. 93 . inoculation were submerged in a 1 mg/ml solution of DAB, pH 3,8 for 8h under constant. 94 . light. Finally leaves were bleached in boiling ethanol (96%) for 10 minutes in order to. 95 . remove the background color of the leaf to only visualize dark brown areas product of. 96 . polymerization in the reaction between H2O2 and DAB. Stained leaves were maintained in. 97 . Petri dishes and photographed.. 5 .

(6) 98 99 . Microscopy. 100 . Detached leaves from inoculated and mock-inoculated plants at 0, 24, 48, 72 and 96. 101 . hour post inoculation were kept in a humid chamber in Petri dishes. Leaves were observed. 102 . directly by low vacuum electron microscopy with a JEOL JSM 6490-LV scanning electron. 103 . microscope. Small samples of tissue at the same times were examined under light. 104 . microscopy and stereomicroscope in order to identify structures of the pathogen on the. 105 . leaflet surface.. 106 107 . Determination of enzymatic activities. 108 . We performed an enzymatic activity assay of three PR proteins Peroxidase,. 109 . Phenylalanine ammonia-lyase and β-1,3 Glucanase. Freshly cut cape gooseberry leaves. 110 . from 0, 6, 12, 24, 48, 72 and 96 hours post inoculation and 6, 9 and 12 days post. 111 . inoculation were frozen in liquid N2 and ground to a fine powder. Samples were stored at -. 112 . 80oC until needed.. 113 . Phenylalanine ammonia-lyase activity assay was performed using L- Phenylalanine. 114 . as a substrate and measuring the amount of trans-Cinnamic acid produced, according to the. 115 . method described by (Thangavelu, Palaniswami et al. 2003). Peroxide activity assay was. 116 . performed colorimetrically using guaiacol as a substrate, following the method described. 117 . by (Malolepsza 2006). Changes in optical densities were determined by measuring. 118 . absorbance at 480 nm for four minutes at 1 minute intervals.. 6 .

(7) 119 . β-1,3 Glucanase activity assay was performed following the method described by. 120 . (Thangavelu, Palaniswami et al. 2003) measuring the production of glucose from the β-1,3. 121 . glucan Laminarin (Sigma-Aldrich Inc., St. Louis, MO) at 500nm. The concentration of. 122 . glucose in the assays was calculated using a standard curve with known concentrations of. 123 . glucose starting at 1mg/ml and four 1/1 serial dilutions.. 124 . Total protein concentrations of all the extracts used for enzymatic activities were. 125 . determined by the Bradford assay (Bradford 1976), using Bovine Serum Albumin as. 126 . Standard (BSA) (Sigma-Aldrich Inc., St. Louis, MO). Enzymatic activity was calculated as. 127 . the product formed in each reaction divided by the total protein concentration of each. 128 . extract (µM product /mg of total protein).. 129 130 . RNA extraction and primer design.. 131 . Frozen plant tissues were ground, using a cold mortar and pestle. The Cape. 132 . gooseberry RNA was then extracted using the ConcertTM Plant RNA Reagent Kit from. 133 . Invitrogen following the manufacturer’s instructions. The presence and the quality of the. 134 . RNA extracted were confirmed by running samples in a formaldehyde denaturing gel at. 135 . 100V for 1 hour. cDNA was generated from total RNA using the iScript Select cDNA. 136 . synthesis kit (BIORAD, Hercules, CA) following the manufacturer’s instructions. GLUA. 137 . (Acidic glucanase) gene was chosen as marker for the Salicilyc acid pathway and CA. 138 . (Carbonic anhydrase) and GT (Glutathione reductase) were used as markers for the. 139 . antioxidative system. Primer 3 (Rozen and Skaletsky 2000) was used to design the primers. 140 . 5’-AAGAATGGCAAGAAGGAGCA-3’ and 5’-GCTTGCTACGACTCCTGGTC-3 for 7 .

(8) 141 . GLUA, 5’-CTGGCCCTTTGCTAGTTCAC-3. 142 . 3. 143 . GCACGCTTCGGTAACTCTTC-3. 144 . 5’-ACGACCAACAGGGACAGTTC-3’ for Elongation factor EF gene used as a. 145 . constitutive expressed endogenous control.. for. CA,. and 5’-GAAATGGCAACCGAATCCTA-. 5’-AGGGAAGGGTGATAGGTCCA-3. and. 5’-. for GT and 5’-ACCACTGGTGGTTTTGAAGC-3’. 146 147 . Real time PCR. 148 . For the quantitative real time PCR the 2-Step SsoFastEvaGreen Supermix. 149 . (BIORAD, Hercules, CA) Kit was employed. A master mix was prepared for each of the. 150 . primers used in this study following the manufacturer’s instructions, adding 1µL of total. 151 . cDNA. qRT-PCR was run at an iCycler iQ5Tm from BIORAD (Hercules, CA) with the. 152 . following conditions: 1 initial denaturation cycle at 95°C for 30 sec to activate the hot start. 153 . DNA polymerase and to denature the template cDNA followed by 45 cycles of a two-step. 154 . procedure: 5 sec at 95°C, and a final step of 10 sec at 57,7°C. After this, a melting curve. 155 . was performed adding an extra step of 65-95°C increasing 0.5°C every 10 sec. Real time. 156 . PCR reactions were carried out in triplicate in 96-well plates. We also included two non-. 157 . template controls lacking template for each pair of primers tested. A standard curve was. 158 . obtained by mixing 2 ul of cDNA of all of the time points used in our study starting at 1200. 159 . ng and four 1/10 serial dilutions. Reverse transcription qRT-PCR product generated from. 160 . RNA isolated from mock-inoculated plants were used as a reference sample (calibrator).. 161 . One plate was prepared per biological replicate and three biological replicates were. 8 .

(9) 162 . performed. Results were analyzed in the iCycler iQ5TM program and relative expression. 163 . was calculated using REST 2009 Software (Pfaffl, Horgan et al. 2002).. 164 165 . Results. 166 . Microscopic observations.. 167 . The first stages of the interaction between Phytophthora infestans and its host. 168 . Physalis peruviana were followed through a microscopical study. The development of the. 169 . pathogen growing on cape gooseberry leaves was evidenced. Electron micrographs showed. 170 . that at 24 hours post inoculation the zoosporangia of P. infestans had not started to. 171 . germinate on cape gooseberry leaves (Fig 1). It was until 48 hours post inoculation that the. 172 . germinating zoosporangia were evidenced (Fig 1, 2). Light microscopy also showed that at. 173 . 48 hours post inoculation the zoosporangia of the pathogen were able to release zoospores. 174 . on the surface of the leaflet (Fig 3). Interestingly our study also showed that, after 72 hours. 175 . post inoculation, P. infestans was able to develop a considerable amount of aerial mycelia. 176 . on the surface of the leaf but there were no disease symptoms or necrotic areas where the. 177 . pathogen was growing. Zoosporangia collapsed after germination and some of the hyphae. 178 . surrounded the sub-stomatal chamber (data not shown). De novo reproductive structures of. 179 . the pathogen were not observed until the end of the experiment and, macroscopically,. 180 . leaves appeared to be healthy.. 181 182 . 9 .

(10) 183 . In vivo detection of H2O2. 184 . An assay for hydrogen peroxide accumulation was performed in order to determine. 185 . if there was a biochemical defense reaction of cape gooseberry against P. infestans.. 186 . Inoculated leaves and mock inoculated leaves were stained using 3,3 diaminobenzidine. 187 . (DAB). At 0, 12, 24 hours there were no differences between inoculated and mock. 188 . treatments and the production of ROS was not observed. However the presence of reactive. 189 . oxygen species ROS was evidenced with the appearance of dark brown areas in inoculated. 190 . leaves at 48 hour post inoculation (Fig 4 and 5). At 72 hour post inoculation the presence. 191 . of ROS in inoculated leaves was still observed but at 96 h the presence of ROS was not. 192 . different from the mock treatment. The assays were done in three leaves per time point and. 193 . repeated three times showing consistent results. These results suggest that Cape Gooseberry. 194 . plants are mounting a biochemical defense response and that this response is attenuated at. 195 . later time points.. 196 . Protein activity assays. 197 . Since we were able to detect defense reaction mechanisms such as ROS, we. 198 . hypothesized that P. infestans might have an effect on the activity of some PR proteins as. 199 . has been reported before in other P. infestans hosts. Consequently, we performed a protein. 200 . activity assay of three well documented PR proteins β-1,3 Glucanase (GLU), Peroxidase. 201 . (POX) and Phenylalanine ammonia-lyase (PAL). When cape gooseberry plants were. 202 . treated with the P. infestans zoosporangia inoculum, GLU activity was no statistically. 203 . different from the mock treatment at early times post inoculation (Fig 7a). However at 12. 204 . hours post inoculation GLU activity was significantly increased and reached a maximum of. 10 .

(11) 205 . 4 times higher than the control at 24 hours post inoculation (Fig 7a). At 48 hours post-. 206 . inoculation the activity decreased but it was still higher than the mock treatment. Finally at. 207 . later time points post inoculation (6, 9 days) there were no differences with the mock. 208 . treatment.. 209 . The activity of Phenylalaline ammonia lyase (PAL), a key enzyme of the phenylpropanoid. 210 . metabolism, was also measured. Plants sprayed with P. infestans solution did not show. 211 . statistically significant differences compared with the mock treatments at any sampling. 212 . points taken in this study (Fig 7b).. 213 . The time course of Peroxidase (POX) activity was also followed. Similar levels of activity. 214 . were obtained between P. infestans inoculated and mock-treated plants at early times post. 215 . inoculation. At 12 hours there was a significant increase of POX activity and it reached the. 216 . highest value at 24 hours post inoculation in inoculated plants (Fig 7c). After 96 hours POX. 217 . activity in inoculated plants dramatically fell and reached the same concentrations as the. 218 . mock treatment.. 219 220 . Real time PCR. 221 . In order to determine if gene expression of defense pathways and antioxidative. 222 . systems was changing through time in response to the interaction with P. infestans we. 223 . performed a qRT-PCR. We chose acidic glucanase (GluA) as a molecular marker of. 224 . salicylic acid pathway and the plastidic gene carbonic anhidrase (CA) and Gluthatione. 225 . reductase (GT) as molecular markers of antioxidative reactions.. 11 .

(12) 226 . The qRT-PCR results for GluA in inoculated plants evidenced a significant change in gene. 227 . expression at all time points taken in this study compared with the expression levels of. 228 . mock-inoculated plants. At 24 hours post inoculation, GluA was up-regulated showing a. 229 . tenfold increase in its expression. Interestingly, after this time point, the expression levels. 230 . decreased but remained at least 1,5 times higher compared to the control until the end of the. 231 . experiment at 6 days post inoculation.. 232 . The plastidic CA gene experienced the biggest change in expression of all the genes tested. 233 . in this study. Starting at 24 hours, there was a 29 fold significant increase in the expression. 234 . of this gene in inoculated plants compared to the mock-inoculated controls. CA was also. 235 . up regulated at all other time points tested. At 48 hours post inoculation, CA showed an. 236 . expression level at least 40 times higher than the mock-inoculated control and at 72 hours. 237 . post inoculation the CA expression levels remained high with a small decrease at the final. 238 . time point (6 days post inoculation). Finally the qRT-PCR results for Glutathione reductase. 239 . showed no significant change in gene expression for the inoculated plants compared to the. 240 . mock treatment at any of the time points during our experiment.. 241 242 . Discussion. 243 . While the interaction between P. infestans and hosts such as potato and tomato is. 244 . well documented, little is known about the interaction between P. infestans and other. 245 . members of the Solanaceae family. This is the first report of responses triggered by P.. 246 . infestans in cape gooseberry. We demonstrated that the cape gooseberry ecotype used in. 12 .

(13) 247 . this study, the most common one grown in Colombia, showed plant defense responses. 248 . typically detected in a resistant host.. 249 . The Hypersensitive Response (HR) is one of the main defense responses associated. 250 . with all forms of resistance against P. infestans (Vleeshouwers, van Dooijeweert et al.. 251 . 2000). ROS production has been proposed as a marker of HR in plant defense reactions. 252 . (Orozco-Cardenas and Ryan 1999) and it is well known as a response after successful. 253 . recognition of plant pathogens (Torres and Dangl 2005). In our study we evidenced the. 254 . accumulation of ROS in hypersensitive response type lesions after 48 hours post. 255 . inoculation. Interestingly, the time when ROS accumulation was first detected matches. 256 . with the germination of the majority of zoosporangia observed in the microscopic study.. 257 . This suggested that the production of ROS could be triggered by the pathogen structures. 258 . when it is trying to penetrate into the host cells. This evidence, in addition to the lack of. 259 . disease symptoms in our assays, suggest that recognition of P. infestans by cape gooseberry. 260 . plants takes place as an important barrier in order to avoid the disease outcome in this. 261 . interaction.. 262 . An increase in PR proteins such as Peroxidase POX has been correlated with. 263 . resistance in different interactions through a variety of mechanisms (Lagrimini 1991)(De. 264 . Ascensao and Dubery 2000). POX has been associated with the last enzymatic step of. 265 . enzyme biosynthesis, the oxidation of hydroxyl cinnamyl alcohols into free radical. 266 . intermediates which then are coupled into the lignin polymer. PAL, the main enzyme in the. 267 . phenylpropanoid metabolism biosynthesis pathway, supplies the carbon skeletons for. 268 . secondary products such as phenolics, which are the precursor molecules for lignin. In our. 269 . assays the activity of this enzyme was not different from the controls therefore the increase 13 .

(14) 270 . in POX activity seems to have a small effect in the lignin biosynthesis pathway. POX. 271 . activity has been found to inhibit the growth of several pathogens and positively correlates. 272 . with resistance to pathogens. (Peng and Kuc 1992; Joseph, Koon et al. 1998; Thangavelu,. 273 . Palaniswami et al. 2003). It has also been implicated in ROS generation as well as ROS. 274 . removal (Wojtaszek 1997; Malolepsza 2006). Our combined results with the increase in. 275 . ROS generation and increase in POX activity suggest that probably both act as a key. 276 . element of cape gooseberry defense system against P. infestans, maybe by restricting the. 277 . development of the pathogen in the host cells creating an unfavorable environment for the. 278 . hyphae.. 279 . An increment in β-1,3 Glucanase activity has been reported for potato and tomato as. 280 . one of the major PR proteins responses against P. infestans (Kombrink, Schröder et al.. 281 . 1988; Schröder, Hahlbrock et al. 1992). In spite of their wide use as markers for defense. 282 . responses, little is known about its mechanism of action and its role is not very clear. It has. 283 . been proposed that the main target for this hydrolytic enzymes are cell wall components of. 284 . the pathogen (Schröder, Hahlbrock et al. 1992). In potato, glucanase activities seem to. 285 . appear at latter stages of the infection and seem to be increased only in compatible. 286 . interactions. Apparently, they do not play a main role in race-cultivar specific resistance,. 287 . being slowly and systemically activated (Schröder, Hahlbrock et al. 1992). In our study the. 288 . activity of GLU in cape gooseberry showed a different trend from that reported in potato. 289 . and seems to have a more important role in the defense response against P. infestans.. 290 . The differential expression of the salicylic acid marker and the oxidative system. 291 . marker was interesting. The up-regulation of the plastidic gene for the carbonic anhydrase. 292 . CA at all points of sampling suggests that there is an increase in the oxidative pathway 14 .

(15) 293 . when cape gooseberry plants are challenged with P. infestans. Some studies have reported. 294 . the CA up-regulation at early time points during the incompatible interaction between P.. 295 . infestans and potato or Nicotiana benthamiana, contrasting with the down-regulation. 296 . during the compatible interaction between the same hosts (Slaymaker, Navarre et al. 2002;. 297 . Restrepo, Myers et al. 2005). In fact plastidic carbonic anhydrase seems to have a key role. 298 . in the activation of HR in resistant genotypes against P.infestans in potato (Restrepo, Myers. 299 . et al. 2005). CA is located in the stroma of the chloroplast and has been shown to facilitate. 300 . the supply of CO2 to Rubisco by maintaining equilibrium between HCO and CO2 within. 301 . the chloroplast and it had been demonstrated to bind Salicylic acid, but it seems that those. 302 . two functions are independent. Further research is needed in order to know how CA. 303 . regulates the ROS levels inside of the chloroplast and in which ways SA interacts with CA. 304 . in the HR.. 305 . It is interesting that GluA, which has been shown to be induced by SA (Gu, Yang et. 306 . al. 2000) was up-regulated in all time points of our experiment. This result suggests that. 307 . salicylic acid might play an important role in the activation of the defense mechanisms. 308 . caused by P. infestans in cape gooseberry. The fact that CA is able to bind with salicylic. 309 . acid and both of them are up regulated in our experiment made us hypothesize that maybe. 310 . they are working together as a part of a defense mechanism. Our results also agree with. 311 . studies done in other hosts and identify the chloroplast as an important location in the plant. 312 . defense response against pathogens such as P. infestans. It is well known that the main. 313 . consequences of the disease are the down-regulation of photosynthesis related genes and. 314 . changes in photosynthetic electron transport (Schnabel, Strittmatter et al. 1998; Restrepo,. 15 .

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(20) Figure 1. Electron microscopy photographs taken on cape gooseberry leaves inoculated with a suspension of P. infestans zoosporangia. A. 24 HPI encysted P. infestans zoospore near to a glandular trichome. B. 48 HPI Germinating encysted zoospore and a germ tube growing in the surface of the leaflet. C and D 72 and 96 HPI active aerial mycelial growing. (HPI: hours post-inoculation). Figure 2. Light microscopy photographs. Left, germinating zoosporangia of Phytophthora infestans on a cape gooseberry leaf at 48 HPI. Right, Zoosporangia and the development of aerial mycelium of P. infestans on a cape gooseberry leaf. (HPI: Hours Post Inoculation). Figure 3. Light microscopy photograph. Zoosporangia of P. infestans releasing zoospores on the surface of cape gooseberry leaflets. Figure 4. Diaminobenzidine staining of cape gooseberry leaves inoculated at different times post inoculation. The presence of hydrogen peroxide was evidenced at later stages of the assay. Since 48 hours post inoculation brown areas could be seen as a result of an increase in the quantity of hydrogen peroxide. A. inoculated leaf at 3HPI, B. water inoculated leaf at 3HPI, C. inoculated leaf at 48 HPI, D water inoculated leaf at 48 HPI, D. inoculated leaf at 72 HPI and E. water inoculated leaf at 72 HPI. (HPI: Hours post inoculation). Figure 5. Left, cape gooseberry leaf inoculated with P. infestans. Right: mock treatment at 48 HPI. Figure 6. Protein activity assays of three pathogen related proteins under controlled conditions in cape goosberry. A, Peroxidase. B, Phenylalanine ammonia lyase and C, β-1,3 Glucanase. Figure 7. Change in the expression over the time in A. Acidic glucanase GLUA, B. Carbonic anhydrase CA and C. Glutathione reductase GT. Numbers on the x-axis represent the collection dates following the two treatments. Numbers in the y-axis represent the change in relative expression compared to a mock. Bars represent the expression of each gene studied. Error bars represent the standard deviation..

(21) Figure 1.

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(26) 10 9 8 7 6 5 4 3 2 1 0. A. Inoculated Mock. 0h. 6h 12h 24h 48h 72h 96h 6D 9D 12h. 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0. B. Inoculated Mock. 0h 6h 12h 24h 48h 72h 96h 6D 9D 12D 2,5. C. 2 1,5 Inoculated 1. Mock. 0,5 0 0h 3h 6h 9h 12h 24h 48h 72h 6D 9D 12D. Figure 6..

(27) 100. B. 10. 1 1. 2. 3. 6. 1,4. C. 1,2 1 0,8 0,6 0,4 0,2 0. 1. Figure 7.. 2. 3. 6.

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