Study on citrus response to huanglongbing highlights a down-regulation of defense-related proteins in lemon plants upon 'Ca. Liberibacter asiaticus' infection.

Citrus huanglongbing (HLB) is a highly destructive disease of citrus presumably caused by 'Candidatus Liberibacterasiaticus' (Las), a gram-negative, insect-transmitted, phloem-limited α-proteobacterium. Although almost all citrus plants are susceptible to HLB, reports have shown reduced susceptibility to Las infection in lemon (Citrus limon) plants. The aim of this study is to identify intra-species specific molecular mechanisms associated with Las-induced responses in lemon plants. To achieve this, comparative 2-DE and mass spectrometry, in addition to Inductively Coupled Plasma Spectroscopy (ICPS) analyses, were applied to investigate differences in protein accumulation and the concentrations of cationic elements in leaves of healthy and Las-infected lemon plants. Results showed a differential accumulation of 27 proteins, including an increase in accumulation of starch synthase but decrease in the production of photosynthesis-related proteins in Las-infected lemon plants compared to healthy plants. Furthermore, there was a 6% increase (P > 0.05) in K concentration in leaves of lemon plants upon Las infection, which support results from previous studies and might represent a common response pattern of citrus plants to Las infection. Interestingly, contrary to reports from prior studies, this study showed a general reduction in the production of defense-related pathogen-response proteins but a 128% increase in Zn concentration in lemon plants in response to Las infection. Taken together, this study sheds light on general and intra-species specific responses associated with the response of citrus plants to Las

Differentially produced protein spots from 2-DE analysis of total leaf proteins from healthy or Las-infected lemon plants.

<p>Panels A-M show magnified views of protein spots in representative 2-DE gels containing separated total proteins from leaves of healthy or Las-infected lemon plants. Labeled spots showed significant changes and correspond to the spots presented in in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067442#pone-0067442-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067442#tab2" target="_blank">Tables 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067442#tab3" target="_blank">3</a>. Two-year old healthy plants were either graft-inoculated with side shoots from PCR-confirmed Las-infected bud sticks or uninoculated and leaf samples were analyzed at six months post-inoculation. A sum of 200 µg of total protein was separated according to charge on a pH 4-7 IpG strip and according to mass on 8-16% gradient SDS-polyacrylamide Tris-HCl gels. Protein spots were visualized by staining with Coomassie Brilliant Blue (CBB). <i>M</i>_r, relative molecular mass; pI, isoelectric point.</p

Categorization of differentially produced proteins in lemon plants in response to Las infection.

<p>(A) Venn diagram showing the number of protein spots that showed higher-accumulation (▲) or lower-accumulation (▼) in infected lemon plants compared to healthy plants. (B) Functional category distribution of differentially produced protein spots from comparing 2-DE gel images of the total leaf proteome of healthy or Las-infected lemon plants.</p

PDQuest-generated master gel image showing the general spot pattern of matched protein spots from the total leaf proteome of healthy or Las-infected lemon plants.

<p>Labeled spots were differentially produced in response to Las-infection and described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067442#tab1" target="_blank">Table 1</a>. A sum of 200 µg of total protein was separated according to charge on a pH 4-7 IpG strip and according to mass on 8-16% gradient SDS-polyacrylamide Tris-HCl gels. Protein spots were visualized by staining with Coomassie Brilliant Blue (CBB). <i>M</i>_r, relative molecular mass; pI, isoelectric point.</p

The leaf-nutrient concentrations of healthy or Las-infected lemon plants.

<p> (A) Macronutrients: calcium (Ca), potassium (K) and magnesium (Mg); (B) Micronutrients: iron (Fe), manganese (Mn), zinc (Zn), and copper (Cu). Two-year old healthy plants were either graft-inoculated or uninoculated with PCR-confirmed Las-infected bud sticks and leaf samples were analyzed 6 months post-inoculation. Bars within a plant group with the same lower case letter are not significantly different from each other (<i>P</i> > 0.05).</p

Effect of Ethanol on Differential Protein Production and Expression of Potential Virulence Functions in the Opportunistic Pathogen <em>Acinetobacter baumannii</em>

<div><p><em>Acinetobacter baumannii</em> persists in the medical environment and causes severe human nosocomial infections. Previous studies showed that low-level ethanol exposure increases the virulence of <em>A. baumannii</em> ATCC 17978. To better understand the mechanisms involved in this response, 2-D gel electrophoresis combined with mass spectrometry was used to investigate differential protein production in bacteria cultured in the presence or absence of ethanol. This approach showed that the presence of ethanol significantly induces and represses the production of 22 and 12 proteins, respectively. Although over 25% of the ethanol-induced proteins were stress-response related, the overall bacterial viability was uncompromised when cultured under these conditions. Production of proteins involved in lipid and carbohydrate anabolism was increased in the presence of ethanol, a response that correlates with increased carbohydrate biofilm content, enhanced biofilm formation on abiotic surfaces and decrease bacterial motility on semi-solid surfaces. The presence of ethanol also induced the acidification of bacterial cultures and the production of indole-3-acetic acid (IAA), a ubiquitous plant hormone that signals bacterial stress-tolerance and promotes plant-bacteria interactions. These responses could be responsible for the significantly enhanced virulence of <em>A. baumannii</em> ATCC 17978 cells cultured in the presence of ethanol when tested with the <em>Galleria mellonella</em> experimental infection model. Taken together, these observations provide new insights into the effect of ethanol in bacterial virulence. This alcohol predisposes the human host to infections by <em>A. baumannii</em> and could favor the survival and adaptation of this pathogen to medical settings and adverse host environments.</p> </div

Effect of ethanol on the pH of <i>A. baumannii</i> ATCC 17978 cultures.

<p>Sterile and inoculated LB broth cultures supplemented with 0%, 1%, or 2% ethanol were incubated in an orbital shaker overnight at 37°C. The pH of the sterile samples and cultured supernatants obtained after centrifugation at 10,000 rpm for 10 min was determined immediately. Error bars represent 1 standard error. Horizontal bars with the asterisk (*) symbol identify samples with statistically significant differences (<i>P</i><0.05) in pH values.</p

Effect of ethanol and/or tryptophan on IAA production.

<p>(A) The concentration of IAA in LB or SB <i>A. baumannii</i> ATCC 17978 cultures supplemented with 0%, 1% or 2% ethanol was determined after 24 h incubation at 37°C. (B) The effect of 5 mM L-tryptophan and/or 1% ethanol supplementation on the production of IAA bacteria cultured in SB after 24 h incubation 37°C. The plus (+) and minus (−) signs denote the addition or not of ethanol or tryptophan. IAA concentration values were normalized to an OD₆₀₀ of 1.0 of each tested sample. Error bars represent 1 standard error. Horizontal bars with the asterisk (*) symbol identify samples with statistically significant differences (<i>P</i><0.05) in IAA concentration values. All differences in IAA concentrations shown in panel B are statistically significant (<i>P</i><0.05).</p

2D-polyacrylamide gel electrophoresis of <i>A. baumannii</i> ATCC 17978 whole-cell proteins differentially produced in response to ethanol.

<p>Representative PDQuest-generated master gel image showing the general pattern of matched protein spots isolated from bacteria grown in LB broth containing 0%, 1%, or 2% ethanol. Differentially expressed spots are numbered. <i>M</i>_r, relative molecular weight; p<i>I</i>, isoelectric point. Although spots numbers range from 1 to 56, the spot numbering is not serialized and missing numbers represent proteins the production of which was not affected by the presence of 1% or 2% ethanol and were used for purposes different from those of this study.</p

Differential production of energy and general metabolism-related proteins in response to ethanol.

<p>Table legends same as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051936#pone-0051936-t001" target="_blank">Table 1</a>.</p