Complexity of Brassica oleracea-Alternaria brassicicola Susceptible Interaction Reveals Downregulation of Photosynthesis at Ultrastructural, Transcriptional, and Physiological Levels.

Black spot disease, caused by Alternaria brassicicola in Brassica species, is one of the most devastating diseases all over the world, especially since there is no known fully resistant Brassica cultivar. In this study, the visualization of black spot disease development on Brassica oleracea var. capitata f. alba (white cabbage) leaves and subsequent ultrastructural, molecular and physiological investigations were conducted. Inter- and intracellular hyphae growth within leaf tissues led to the loss of host cell integrity and various levels of organelle disintegration. Severe symptoms of chloroplast damage included the degeneration of chloroplast envelope and grana, and the loss of electron denseness by stroma at the advanced stage of infection. Transcriptional profiling of infected leaves revealed that photosynthesis was the most negatively regulated biological process. However, in infected leaves, chlorophyll and carotenoid content did not decrease until 48 hpi, and several chlorophyll a fluorescence parameters, such as photosystem II quantum yield (Fv/Fm), non-photochemical quenching (NPQ), or plant vitality parameter (Rdf) decreased significantly at 24 and 48 hpi compared to control leaves. Our results indicate that the initial stages of interaction between B. oleracea and A. brassicicola are not uniform within an inoculation site and show a complexity of host responses and fungal attempts to overcome host cell defense mechanisms. The downregulation of photosynthesis at the early stage of this susceptible interaction suggests that it may be a part of a host defense strategy, or, alternatively, that chloroplasts are targets for the unknown virulence factor(s) of A. brassicicola. However, the observed decrease of photosynthetic efficiency at the later stages of infection is a result of the fungus-induced necrotic lesion expansion.

VpSTS29/STS2 enhances fungal tolerance in grapevine through a positive feedback loop.

Accumulation of stilbene phytoalexins stimulates resistance mechanisms against the grapevine fungus Uncinula necator. However, the defensive mechanisms triggered by stilbene synthase (STS) genes, remain largely unknown. Here, we report the function and molecular mechanism of the stilbene synthase gene VpSTS29/STS2 from Vitis pseudoreticulata in the regulation of plant responses to powdery mildew. Stilbene synthesis occurred mainly in root tips and mesophyll cells of transgenic grapevines via transport through the vascular bundles. Overexpression of VpSTS29/STS2 in Vitis vinifera increased the abundance of STSs in mesophyll tissue and resulted in the accumulation of biologically active resveratrol derivatives at the invasion site. Similarly, expression of VpSTS29/STS2 in Arabidopsis increased resistance to Golovinomyces cichoracearum. The VpSTS29/STS2-expressing Arabidopsis lines showed increased piceid accumulation together with more local hypersensitive reactions, inhibition of mycelial growth, and a reduced incidence of pathogens. Transcriptome profiling analyses demonstrated that VpSTS29/STS2-induced defences led to reprograming of global gene expression and activation of salicylic acid (SA) signalling, thus increasing expression of WRKY-MYB transcription factors and other defence response genes. We propose a model for resveratrol-mediated coordination of defence responses in which SA participates in a positive feedback loop.

Inhibition of OsSWEET11 function in mesophyll cells improves resistance of rice to sheath blight disease.

Pathogen-host interaction is a complicated process; pathogens mainly infect host plants to acquire nutrients, especially sugars. Rhizoctonia solani, the causative agent of sheath blight disease, is a major pathogen of rice. However, it is not known how this pathogen obtains sugar from rice plants. In this study, we found that the rice sugar transporter OsSWEET11 is involved in the pathogenesis of sheath blight disease. Quantitative real-time polymerase chain reaction (qRT-PCR) and ß-d-glucuronidase expression analyses showed that R. solani infection significantly enhanced OsSWEET11 expression in leaves amongst the clade III SWEET members. The analyses of transgenic plants revealed that Ossweet11 mutants were less susceptible, whereas plants overexpressing OsSWEET11 were more susceptible, to sheath blight compared with wild-type controls, but the yield of OsSWEET11 mutants and overexpressors was reduced. SWEETs become active on oligomerization. Split-ubiquitin yeast two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays showed that mutated OsSWEET11 interacted with normal OsSWEET11. In addition, expression of conserved residue mutated AtSWEET1 inhibited normal AtSWEET1 activity. To analyse whether inhibition of OsSWEET11 function in mesophyll cells is related to defence against this disease, mutated OsSWEET11 was expressed under the control of the Rubisco promoter, which is specific for green tissues. The resistance of transgenic plants to sheath blight disease, but not other disease, was improved, whereas yield production was not obviously affected. Overall, these results suggest that R. solani might acquire sugar from rice leaves by the activation of OsSWEET11 expression. The plants can be protected from infection by manipulation of the expression of OsSWEET11 without affecting the crop yield.

SnTox1, a Parastagonospora nodorum necrotrophic effector, is a dual-function protein that facilitates infection while protecting from wheat-produced chitinases.

SnTox1 induces programmed cell death and the up-regulation of pathogenesis-related genes including chitinases. Additionally, SnTox1 has structural homology to several plant chitin-binding proteins. Therefore, we evaluated SnTox1 for chitin binding and localization. We transformed an avirulent strain of Parastagonospora nodorum as well as three nonpathogens of wheat (Triticum aestivum), including a necrotrophic pathogen of barley, a hemibiotrophic pathogen of sugar beet and a saprotroph, to evaluate the role of SnTox1 in infection and in protection from wheat chitinases. SnTox1 bound chitin and an SnTox1-green fluorescent fusion protein localized to the mycelial cell wall. Purified SnTox1 induced necrosis in the absence of the pathogen when sprayed on the leaf surface and appeared to remain on the leaf surface while inducing both epidermal and mesophyll cell death. SnTox1 protected the different fungi from chitinase degradation. SnTox1 was sufficient to change the host range of a necrotrophic pathogen but not a hemibiotroph or saprotroph. Collectively, this work shows that SnTox1 probably interacts with a receptor on the outside of the cell to induce cell death to acquire nutrients, but SnTox1 accomplishes a second role in that it protects against one aspect of the defense response, namely the effects of wheat chitinases.

Magnaporthe oryzae effectors MoHEG13 and MoHEG16 interfere with host infection and MoHEG13 counteracts cell death caused by Magnaporthe-NLPs in tobacco.

KEY MESSAGE: Adapted pathogens are able to modulate cell responses of their hosts most likely due to the activity of secreted effector molecules thereby enabling colonisation by ostensible nonhost pathogens. It is postulated that host and nonhost pathogens of a given plant species differ in their repertoire of secreted effector molecules that are able to suppress plant resistance. We pursued the strategy of identifying novel effectors of Magnaporthe oryzae, the causal agent of blast disease, by comparing the infection process of closely related host vs. nonhost Magnaporthe species on barley (Hordeum vulgare L.). When both types of pathogen simultaneously attacked the same cell, the nonhost isolate became a successful pathogen possibly due to potent effectors secreted by the host isolate. Microarray studies led to a set of M. oryzae Hypothetical Effector Genes (MoHEGs) which were classified as Early- and LateMoHEGs according to the maximal transcript abundance during colonization of barley. Interestingly, orthologs of these MoHEGs from a nonhost pathogen were similarly regulated when investigated in a host situation, suggesting evolutionary conserved functions. Knockout mutants of MoHEG16 from the group of EarlyMoHEGs were less virulent on barley and microscopic studies revealed an attenuated transition from epidermal to mesophyll colonization. MoHEG13, a LateMoHEG, was shown to antagonize cell death induced by M. oryzae Necrosis-and ethylene-inducing-protein-1 (Nep1)-like proteins in Nicotiana benthamiana. MoHEG13 has a virulence function as a knockout mutant showed attenuated disease progression when inoculated on barley.

Comparison of cell death and accumulation of reactive oxygen species in wheat lines with or without Yr36 responding to Puccinia striiformis f. sp. tritici under low and high temperatures at seedling and adult-plant stages.

Yr36 is an important gene conferring resistance to stripe rust of wheat caused by Puccinia striiformis f. sp. tritici (Pst). To determine if the Yr36 resistance is correlated to reactive oxygen species (ROS) burst and cell death, wheat near-isogenic lines with (UC1041 + Yr36) and without (UC1041) the gene were histologically characterized for response to Pst infection. Yr36 conferred stripe rust resistance at both seedling and adult-plant stages when the gene line was tested with Pst race CYR29 at a high-temperature (HT) cycle (12 °C at night and 33 °C during the day). At the HT cycle, the growth of secondary hyphae was obviously suppressed in both seedlings and adult plants of UC1041 + Yr36 compared with those of UC1041. The percentages of infection sites with necrotic host cells in UC1041 + Yr36 were significantly higher than UC1041 60 hours after inoculation (hai) at both seedling and adult-plant stages. Mesophyll cell death in the inoculated UC1041 + Yr36 leaves at the HT cycle was stronger than at a low-temperature (LT) cycle (12 °C at night and 18 °C during the day). At the HT cycle, the level of ROS burst started increasing in the inoculated leaves of UC1041 + Yr36 when Pst hyphae started differentiating and extending, and simultaneously, the number of penetration sites with hypersensitive cell death was also increasing. The results indicate that Yr36 product affects the ROS accumulation and cell death of the host in interaction of wheat with Pst.

Veinal-mesophyll interaction under biotic stress.

According to microscopic observations, germinating hyphae of Botrytis cinerea, though easily penetrating Mesembryanthemum crystallinum mesophyll tissue, are limited in growth in mid-ribs and only occasionally reach vascular bundles. In mid-ribs of C3 and CAM leaves, we found significantly lower rbcL (large RubisCO subunit) abundance. Moreover, in CAM leaves, minute transcript contents for pepc1 (phosphoenolpyruvate carboxylase) and nadpme1 (malic enzyme) genes found in the mid-ribs suggest that they perform ß-carboxylation at a low rate. The gene of the main H2O2-scavenging enzyme, catL (catalase), showed lower expression in C3 mid-rib parts in comparison to mesophyll. This allows maintenance of higher H2O2 quantities in mid-rib parts. In C3 leaves, pathogen infection does not impact photosynthesis. However, in CAM plants, the expression profiles of rbcL and nadpme1 were similar under biotic stress, with transcript down-regulation in mid-ribs and up-regulation in mesophyll (however, in case of rbcL not significant). After B. cinerea infection in C3 plants, transcripts for both antioxidative proteins strongly increased in mid-ribs, but not in mesophyll. In infected CAM plants, a significant transcript increase in the mesophyll was parallel to its decrease in the mid-rib region (however, in the case of catL this was not significant). Pathogen infection modified the expression of carbon and ROS metabolism genes in mid-ribs and mesophyll, resulting in the establishment of successful leaf defense.

Water balance altered in cucumber plants infected with Fusarium oxysporum f. sp. cucumerinum.

Fusarium wilt is caused by the infection and growth of the fungus Fusarium oxysporum in the xylem of host plants. The physiological responses of cucumbers that are infected with Fusarium oxysporum f. sp. cucumerinum (FOC) was studied in pot and hydroponic experiments in a greenhouse. The results showed that although water absorption and stem hydraulic conductance decreased markedly in infected plants, large amounts of red ink accumulated in the leaves of infected cucumber plants. The transpiration rate (E) and stomatal conductance (gs) of the infected plants were significantly reduced, but the E/gs was higher than healthy plants. We further found that there was a positive correlation between leaf membrane injury and E/gs, indicating that the leaf cell membrane injury increased the non-stomatal water loss from infected plants. The fusaric acid (FA), which was detected in the infected plant, resulted in damage to the leaf cell membranes and an increase in E/gs, suggesting that FA plays an important role in non-stomatal water loss. In conclusion, leaf cell membrane injury in the soil-borne Fusarium wilt of cucumber plants induced uncontrolled water loss from damaged cells. FA plays a critical role in accelerating the development of Fusarium wilt in cucumber plants.

Effects of slag-based silicon fertilizer on rice growth and brown-spot resistance.

It is well documented that slag-based silicon fertilizers have beneficial effects on the growth and disease resistance of rice. However, their effects vary greatly with sources of slag and are closely related to availability of silicon (Si) in these materials. To date, few researches have been done to compare the differences in plant performance and disease resistance between different slag-based silicon fertilizers applied at the same rate of plant-available Si. In the present study both steel and iron slags were chosen to investigate their effects on rice growth and disease resistance under greenhouse conditions. Both scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to examine the effects of slags on ultrastructural changes in leaves of rice naturally infected by Bipolaris oryaze, the causal agent of brown spot. The results showed that both slag-based Si fertilizers tested significantly increased rice growth and yield, but decreased brown spot incidence, with steel slag showing a stronger effect than iron slag. The results of SEM analysis showed that application of slags led to more pronounced cell silicification in rice leaves, more silica cells, and more pronounced and larger papilla as well. The results of TEM analysis showed that mesophyll cells of slag-untreated rice leaf were disorganized, with colonization of the fungus (Bipolaris oryzae), including chloroplast degradation and cell wall alterations. The application of slag maintained mesophyll cells relatively intact and increased the thickness of silicon layer. It can be concluded that applying slag-based fertilizer to Si-deficient paddy soil is necessary for improving both rice productivity and brown spot resistance. The immobile silicon deposited in host cell walls and papillae sites is the first physical barrier for fungal penetration, while the soluble Si in the cytoplasm enhances physiological or induced resistance to fungal colonization.

The role of autophagy in chloroplast degradation and chlorophagy in immune defenses during Pst DC3000 (AvrRps4) infection.

BACKGROUND: Chlorosis of leaf tissue normally observed during pathogen infection may result from the degradation of chloroplasts. There is a growing evidence to suggest that the chloroplast plays a significant role during pathogen infection. Although most degradation of the organelles and cellular structures in plants is mediated by autophagy, its role in chloroplast catabolism during pathogen infection is largely unknown. RESULTS: In this study, we investigated the function of autophagy in chloroplast degradation during avirulent Pst DC3000 (AvrRps4) infection. We examined the expression of defensive marker genes and suppression of bacterial growth using the electrolyte leakage assay in normal light (N) and low light (L) growing environments of wild-type and atg5-1 plants during pathogen treatment. Stroma-targeted GFP proteins (CT-GFP) were observed with LysoTracker Red (LTR) staining of autophagosome-like structures in the vacuole. The results showed that Arabidopsis expressed a significant number of small GFP-labeled bodies when infected with avirulent Pst DC3000 (AvrRps4). While barely detectable, there were small GFP-labeled bodies in plants with the CT-GFP expressing atg5-1 mutation. The results showed that chloroplast degradation depends on autophagy and this may play an important role in inhibiting pathogen growth. CONCLUSION: Autophagy plays a role in chloroplast degradation in Arabidopsis during avirulent Pst DC3000 (AvrRps4) infection. Autophagy dependent chloroplast degradation may be the primary source of reactive oxygen species (ROS) as well as the pathogen-response signaling molecules that induce the defense response.

UDP-glucosyltransferase UGT84A2/BRT1 is required for Arabidopsis nonhost resistance to the Asian soybean rust pathogen Phakopsora pachyrhizi.

Nonhost resistance (NHR) of plants to fungal pathogens comprises different defense layers. Epidermal penetration resistance of Arabidopsis to Phakopsora pachyrhizi requires functional PEN1, PEN2 and PEN3 genes, whereas post-invasion resistance in the mesophyll depends on the combined functionality of PEN2, PAD4 and SAG101. Other genetic components of Arabidopsis post-invasion mesophyll resistance remain elusive. We performed comparative transcriptional profiling of wild-type, pen2 and pen2 pad4 sag101 mutants after inoculation with P. pachyrhizi to identify a novel trait for mesophyll NHR. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis and microscopic analysis confirmed the essential role of the candidate gene in mesophyll NHR. UDP-glucosyltransferase UGT84A2/bright trichomes 1 (BRT1) is a novel component of Arabidopsis mesophyll NHR to P. pachyrhizi. BRT1 is a putative cytoplasmic enzyme in phenylpropanoid metabolism. BRT1 is specifically induced in pen2 with post-invasion resistance to P. pachyrhizi. Silencing or mutation of BRT1 increased haustoria formation in pen2 mesophyll. Yet, the brt1 mutation did not affect NHR to P. pachyrhizi in wild-type plants. We assign a novel function to BRT1, which is important for post-invasion NHR of Arabidopsis to P. pachyrhizi. BRT1 might serve to confer durable resistance against P. pachyrhizi to soybean.