Comparison of effects on disease symptom development of Arabidopsis Col-0 and its mutant <i>eds</i>16-1, <i>npr</i>1-1 and <i>pad</i>4-1 between zoospore free fluid (ZFF) and zoospores (ZSP) of <i>Phytophthora nicotianae</i>.

<p>Plants were treated with ZFF alone or zoospore suspension at 200,000/ml and recorded symptoms after 88 h at 23°C. A. Percentage of leaves displaying yellowing or rotting after treatment. Each column is a mean of 6 plants; bars depict standard error. Similar results were observed in three independent replicates. B. Images of the plants after treatment. Typical yellowing or rotting is indicated with blue and red arrows, respectively.</p

Effects of zoospore free fluid (ZFF) of <i>Phytophthora nicotianae</i> on expression of salicylic acid (SA)—and jasmonic acid (JA)—associated defense genes in Arabidopsis Col-0 and its mutants.

<p>Plants were flooded with ZFF, SDW or zoospore suspension at 200,000/ml (ZSP) and extracted for RNA at 4, 8, 16, 24 and 48 h after treatment. <i>PR1</i>, <i>PDF1</i>.<i>2</i> expression was analyzed with QRT-PCR in a real time PCR system and the transcript levels or mean value of Ct (threshold cycle) were normalized with reference gene <i>Actin-1</i> in the same samples. The graph depicts the relative expression of the genes in treated plants. Each column is a mean of three PCR replicates from one of two biological experiments with similar results. Bars depict standard error from the replicates.</p

Microscopy of effects of zoospore free fluid (ZFF) on Arabidopsis (Col-0) responses to <i>Phytophthora nicotianae</i> zoospores.

<p>Individual cauline leaves were flooded in ZFF (A, C, E, and G) or sterile distilled water (SDW) (B, D, F and H) containing zoospores at 1,600 /ml and observed at different exposure time. Bars = 50 μm. Letters: h = hypha or hyphae, sd = substance discharge from plant tissues, sp = sporangia, zsp = zoospores.</p

Zoospore exudates from <i>Phytophthora nicotianae</i> affect immune responses in <i>Arabidopsis</i>

<div><p>Zoospore exudates play important roles in promoting zoospore communication, homing and germination during plant infection by <i>Phytophthora</i>. However, it is not clear whether exudates affect plant immunity. Zoospore-free fluid (ZFF) and zoospores of <i>P</i>. <i>nicotianae</i> were investigated comparatively for effects on resistance of <i>Arabidopsis thaliana</i> Col-0 and mutants that affect signaling mediated by salicylic acid (SA) and jasmonic acid (JA): <i>eds16</i> (enhanced disease susceptibility16), <i>pad4</i> (phytoalexin deficient4), and <i>npr1</i> (nonexpressor of pathogenesis-related genes1). Col-0 attracted more zoospores and had severe tissue damage when flooded with a zoospore suspension in ZFF. Mutants treated with ZFF alone developed disease symptoms similar to those inoculated with zoospores and requirements of EDS16 and PAD4 for plant responses to zoospores and the exudates was apparent. Zoospore and ZFFs also induced expression of the <i>PR1</i> and <i>PDF1</i>.<i>2</i> marker genes for defense regulated by SA and JA, respectively. However, ZFF affected more JA defense signaling, down regulating <i>PR</i>1 when SA signaling or synthesis is deficient, which may be responsible for <i>Arabidopsis</i> mutant plants more susceptible to infection by high concentration of <i>P</i>. <i>nicotianae</i> zoospores. These results suggest that zoospore exudates can function as virulence factors and inducers of plant immune responses during plant infection by <i>Phytophthora</i>.</p></div

Difference of effects between zoospore free fluid (ZFF) and zoospores (ZSP) of <i>P</i>. <i>nicotianae</i> on symptom production of <i>Arabidopsis thaliana</i> plants.

<p>Difference of effects between zoospore free fluid (ZFF) and zoospores (ZSP) of <i>P</i>. <i>nicotianae</i> on symptom production of <i>Arabidopsis thaliana</i> plants.</p

Application of alignment-free bioinformatics methods to identify an oomycete protein with structural and functional similarity to the bacterial AvrE effector protein

<div><p>Diverse plant pathogens export effector proteins to reprogram host cells. One of the most challenging goals in the molecular plant-microbe field is to functionally characterize the complex repertoires of effectors secreted by these pathogens. For bacterial pathogens, the predominant class of effectors is delivered to host cells by Type III secretion. For oomycetes, the predominant class of effectors is defined by a signal peptide that mediates secretion from the oomycete and a conserved RxLR motif. Downy mildew pathogens and <i>Phytophthora</i> species maintain hundreds of candidate RxLR effector genes in their genomes. Although no primary sequence similarity is evident between bacterial Type III effectors (T3Es) and oomycete RXLR effectors, some bacterial and oomycete effectors have convergently evolved to target the same host proteins. Such effectors might have evolved domains that are functionally similar but sequence-unrelated. We reasoned that alignment-free bioinformatics approaches could be useful to identify structural similarities between bacterial and oomycete effectors. To test this approach, we used partial least squares regression, alignment-free bioinformatics methods to identify effector proteins from the genome of the oomycete <i>Hyaloperonospora arabidopsidis</i> that are similar to the well-studied AvrE1 effector from <i>Pseudomonas syringae</i>. This approach identified five RxLR proteins with putative structural similarity to AvrE1. We focused on one, HaRxL23, because it is an experimentally validated effector and it is conserved between distantly related oomycetes. Several experiments indicate that HaRxL23 is functionally similar to AvrE1, including the ability to partially rescue an AvrE1 loss-of-function mutant. This study provides an example of how an alignment-free bioinformatics approach can identify functionally similar effector proteins in the absence of primary sequence similarity. This approach could be useful to identify effectors that have convergently evolved regardless of whether the shared host target is known.</p></div

Nine protein candidates identified from <i>Hyaloperonospora arabidopsidis</i> genome by the three methods.

<p>Nine protein candidates identified from <i>Hyaloperonospora arabidopsidis</i> genome by the three methods.</p

Pph 3121 virulence is not enhanced by transgenic HaRxL23 or AvrE.

<p>Plants were infiltrated with a bacterial suspension of 1 X 10⁵ cfu/ml. Bacterial populations were determined at day 0 and day 3 after inoculation. Error bars indicate Standard Error of six independent leaf samples tested at the same time. The experiment was repeated three times with similar results.</p

HaRxL23 rescues the reduced lesion phenotype of the ΔavrE1 strain in tomato Moneymaker plants.

<p>(A) Disease symptoms (lesion production) on tomato cv. <i>Moneymaker</i> plants 8 days after dip inoculation of the indicated strains of <i>Pto</i> DC3000 at 1x10⁸ cfu/ml bacterial culture. (B) Number of lesions (≥0.25mm²) per whole leaf appearing on plants 8 days after dipping inoculation with the respective bacterial strains. Values indicate mean and error bars indicate standard error on at least five whole leaves assayed for each treatment. P-value * < 0.01; t-test comparisons representing significant differences with DC3000; <b>+</b> < 0.01; t-test comparisons representing significant differences with ΔavrE1. This experiment was repeated three times with similar results.</p

A simplified model for crosstalk between the circadian clock and plant innate immunity.

<p>(<b>A</b>) Timing of stomata-dependent and -independent defense in a day. At night, plants might rely more on closed stomata to provide physical constrains to limit pathogen invasion but have relatively lower levels of stomata-independent defense. Once pathogens bypass such constrains (i.e. via infiltration infection in the laboratory), they encounter a plant host that is more susceptible than during the day. During the day, most stomata are wide open. In the presence of pathogens, plants can only transiently reduce stomatal aperture for a few hours (this study and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003370#ppat.1003370-Melotto1" target="_blank">[1]</a>). Thus, during the day plants might depend more on stomata-independent defense to restrict pathogen invasion. Stomata-dependent defense could be stronger at night while stomata-independent defense could be stronger during the day. (<b>B</b>) The circadian clock regulates both stomata-dependent and -independent defense pathways to restrict pathogen growth in Arabidopsis. In a stomata-dependent pathway, CCA1 and LHY act, at least in part, through GRP7 as a direct downstream target to regulate stomatal aperture and thereby defense. Other downstream targets of CCA1 and LHY and other components of the central oscillator of the circadian clock might also be involved in regulating stomata-dependent and –independent defense. On the other hand, pathogen infection can activate PTI, ETI and other defense signaling in the host. PTI induced by flg22 feeds back to regulate clock activity. In addition, flg22-triggered signaling is under circadian clock control <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003370#ppat.1003370-Bhardwaj1" target="_blank">[27]</a>. Thus, we conclude that the clock-defense crosstalk involves flg22-mediated signaling. Flg22 can affect stomatal aperture <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003370#ppat.1003370-Zhang1" target="_blank">[91]</a>. However, whether this function of flg22 is through its regulation of the circadian clock or through a direct regulation of stomata is unclear. Other questions, such as whether additional PAMPs, effectors, and other defense signaling molecules are involved in clock-defense crosstalk, remain to be answered.</p