Mutation of the Glucosinolate Biosynthesis Enzyme Cytochrome P450 83A1 Monooxygenase Increases Camalexin Accumulation and Powdery Mildew Resistance

Small secondary metabolites, including glucosinolates and the major phytoalexin camalexin, play important roles in immunity in Arabidopsis thaliana. We isolated an Arabidopsis mutant with increased resistance to the powdery mildew fungus Golovinomyces cichoracearum and identified a mutation in the gene encoding cytochrome P450 83A1 monooxygenase (CYP83A1), which functions in glucosinolate biosynthesis. The cyp83a1-3 mutant exhibited enhanced defense responses to G. cichoracearum and double mutant analysis showed that this enhanced resistance requires NPR1, EDS1, and PAD4, but not SID2 or EDS5. In cyp83a1-3 mutants, the expression of genes related to camalexin synthesis increased upon G. cichoracearum infection. Significantly, the cyp83a1-3 mutant also accumulated higher levels of camalexin. Decreasing camalexin levels by mutation of the camalexin synthetase gene PAD3 or the camalexin synthesis regulator AtWRKY33 compromised the powdery mildew resistance in these mutants. Consistent with these observations, overexpression of PAD3 increased camalexin levels and enhanced resistance to G. cichoracearum. Taken together, our data indicate that accumulation of higher levels of camalexin contributes to increased resistance to powdery mildew

Origin, evolution and diversification of plant ARGONAUTE proteins

Argonaute (AGO) proteins are central players in RNA interference in eukaryotes. They associate with small RNAs (sRNA) and lead to transcriptional or posttranscriptional silencing of targets, thereby regulating diverse biological processes. The molecular and biological functions of AGO proteins have been extensively characterized, particularly in a few angiosperm species, leading to the recognition that the AGO family has expanded to accommodate diverse sRNAs thereby performing diverse biological functions. However, understanding of the expansion of AGO proteins in plants is still limited, due to a dearth of knowledge of AGO proteins in green algal groups. Here, we identified more than 2900 AGO proteins from 244 plant species, including green algae, and performed a large-scale phylogenetic analysis. The phylogeny shows that the plant AGO family gave rise to four clades after the emergence of hydrobiontic algae and prior to the emergence of land plants. Subsequent parallel expansion in ferns and angiosperms resulted in eight main clades in angiosperms: AGO2, AGO7, AGO6, AGO4, AGO1, AGO10a, AGO10b and AGO5. On the basis of this phylogeny, we identified two novel AGO4 orthologs that Arabidopsis does not have, and redefined AGO10, which is composed of AGO10a and AGO10b. Finally, we propose a hypothetical evolutionary model of AGO proteins in plants. Our studies provide a deeper understanding of the phylogenetic relationships of AGO family members in the green lineage, which would help to further reveal their roles as RNAi effectors

The Effect of Phosphorylation on the Salt-tolerance-related Functions of the Soybean Protein PM18, a Member of the Group-3 LEA Protein Family

Enzymatically driven post-translated modifications (PTMs) usually happen within the intrinsically disordered regions of a target protein and can modulate variety of protein functions. Late embryogenesis abundant (LEA) proteins are a family of the plant intrinsically disordered proteins (IDPs). Despite their important roles in plant stress response, there is currently limited knowledge on the presence and functional and structural effects of phosphorylation on LEA proteins. In this study, we identified three phosphorylation sites (Ser90, Tyr136, and Thr266) in the soybean PM18 protein that belongs to the group-3 LEA proteins. In yeast expression system, PM18 protein increased the salt tolerance of yeast, and the phosphorylation of this protein further enhanced its protective function. Further analysis revealed that Ser90 and Tyr136 are more important than Thr266, and these two sites might work cooperatively in regulating the salt resistance function of PM18. The circular dichroism analysis showed that PM18 protein was disordered in aqueous media, and phosphorylation did not affect the disordered status of this protein. However, phosphorylation promoted formation of more helical structure in the presence of sodium dodecyl sulfate (SDS) or trifluoroethanol (TFE). Furthermore, in dedicated in vitro experiments, phosphorylated PM18 protein was able to better protect lactate dehydrogenase (LDH) from the inactivation induced by the freeze-thaw cycles than its un- or dephosphorylated forms. All these data indicate that phosphorylation may have regulatory effects on the stress-tolerance-related function of LEA proteins. Therefore, further studies are needed to shed more light on functional and structural roles of phosphorylation in LEA proteins

Intrinsically Disordered Proteins as Important Players During Desiccation Stress of Soybean Radicles

Intrinsically disordered proteins (IDPs) play a variety of important physiological roles in all living organisms. However, there is no comprehensive analysis of the abundance of IDPs associated with environmental stress in plants. Here, we show that a set of heat-stable proteins (i.e., proteins that do not denature after boiling at 100 °C for 10 min) was present in R0mm and R15mm radicles (i.e., before radicle emergence and 15 mm long radicles) of soybean (Glycine max) seeds. This set of 795 iTRAQ-quantified heat-stable proteins contained a high proportion of wholly or highly disordered proteins (15%), which was significantly higher than that estimated for the whole soybean proteome containing 55,787 proteins (9%). The heat-stable proteome of soybean radicles that contain many IDPs could protect lactate dehydrogenase (LDH) during freeze–thaw cycles. Comparison of the 795 heat-stable proteins in the R0mm and R15mm soybean radicles revealed that many of these proteins changed abundance during seedling growth with 170 and 89 proteins being more abundant in R0mm and R15mm, respectively. KEGG analysis identified 18 proteins from the cysteine and methionine metabolism pathways and nine proteins from the phenylpropanoid biosynthesis pathway. As an important type of IDP related to stress, 30 late embryogenesis abundant proteins were also found. Ten selected proteins with high levels of predicted intrinsic disorder were able to efficiently protect LDH from the freeze–thaw-induced inactivation, but the protective ability was not correlated with the disorder content of these proteins. These observations suggest that protection of the enzymes and other proteins in a stressed cell can be one of the biological functions of plant IDPs

Blocking the Thiol at Cysteine-322 Destabilizes Tau Protein and Prevents Its Oligomer Formation

Abnormal accumulation of tau protein into oligomers contributes to neuronal dysfunction. Reduction of tau level is potentially able to prevent its accumulation. Here we uncover a critical role of the free thiol at Cys-322 in determining tau stability. We found that the application of thiol-blocking agents like NEM or MMTS blocks this thiol, by which it destabilizes tau protein and prevents its oligomer formation. Furthermore, we identified a tau-interacting protein, selenoprotein W, which attenuates tau accumulation by forming disulfide linkage between SelW Cys-37 and tau Cys-322. These findings provide a promising strategy to prevent tau accumulation and oligomer formation

A Truncated NLR Protein, TIR-NBS2, Is Required for Activated Defense Responses in the <i>exo70B1</i> Mutant

<div><p>During exocytosis, the evolutionarily conserved exocyst complex tethers Golgi-derived vesicles to the target plasma membrane, a critical function for secretory pathways. Here we show that <i>exo70B1</i> loss-of-function mutants express activated defense responses upon infection and express enhanced resistance to fungal, oomycete and bacterial pathogens. In a screen for mutants that suppress <i>exo70B1</i> resistance, we identified nine alleles of <i>TIR-NBS2</i> (<i>TN2</i>), suggesting that loss-of-function of <i>EXO70B1</i> leads to activation of this nucleotide binding domain and leucine-rich repeat-containing (NLR)-like disease resistance protein. This NLR-like protein is atypical because it lacks the LRR domain common in typical NLR receptors. In addition, we show that TN2 interacts with EXO70B1 in yeast and <i>in planta</i>. Our study thus provides a link between the exocyst complex and the function of a ‘TIR-NBS only’ immune receptor like protein. Our data are consistent with a speculative model wherein pathogen effectors could evolve to target EXO70B1 to manipulate plant secretion machinery. TN2 could monitor EXO70B1 integrity as part of an immune receptor complex.</p></div

<i>exo70B1-3</i> mutants display enhanced resistance to <i>H.a.</i> Noco2, <i>Pto</i> DC3000 and <i>Pto</i> DC3000 <i>avrRpt2</i>.

<p><b>(A)</b> Two-week-old seedlings were inoculated with <i>H.a.</i> Noco2. The number of spores was counted at 7 dpi. The asterisk indicates a significant difference from WT (<i>p <</i> 0.01; Student’s <i>t</i>-test). <b>(B)-(C)</b> Four-week-old plants were infiltrated with <i>Pto</i> DC3000 <b>(B)</b> and <i>Pto</i> DC3000 <i>avrRpt2</i><b>(C)</b>. Bacterial growth was monitored at 0 and 3 dpi. cfu: colony-forming units. The asterisk indicates a significant difference from wild type (<i>p <</i> 0.01; Student’s <i>t</i>-test). Bars represent mean and standard deviation of values from three biological samples. The experiments were repeated three times with similar results.</p

<i>exo70B1-3</i> mutants display enhanced resistance to <i>G. cichoracearum</i>.

<div><p><b>(A)</b> Plants were grown in the standard short day conditions. Upper three panels: Uninfected four-, five- and six-week-old wild type and <i>exo70B1-3</i> plants were photographed. <i>exo70B1-3</i> mutants started to develop spontaneous cell death at five weeks of age, and cell death was more pronounced at six weeks of age. Lower two panels: Four-week-old plants were infected with <i>G. cichoracearum</i> and the plants or representative leaves were photographed at 7 dpi. The wild type plants displayed a large number of fungal spores on the leaves, but the <i>exo70B1-3</i> mutants displayed very few spores, with massive necrotic lesions on the leaves. Five-week-old wild type and <i>exo70B1-3</i> plants were the uninoculated controls for plants infected with <i>G. cichoracearum</i>. Bar = 20 mm.</p> <p><b>(B)</b> Infected leaves at 7 dpi were stained with trypan blue to show the fungal structures and dead cells (arrows). Bar = 100 μm.</p> <p><b>(C)</b> Fungal growth in plants was assessed by counting the number of conidiophores per colony at 5 dpi. The asterisk indicates a significant difference from wild type (<i>p <</i> 0.01; Student’s <i>t</i>-test). Bars represent mean and standard deviation (n>30). The experiments were repeated three times with similar results.</p> <p><b>(D)</b> Infected leaves were stained with aniline blue at 2 dpi to examine callose deposition (blue dots, indicated by red quadrangle). Bar = 50 μm.</p> <p><b>(E)</b> Infected leaves were stained with 3,3′-diamino benzidine-HCl (DAB) at 2 dpi to visualize accumulation of hydrogen peroxide (brown staining, indicated by arrow). Bar = 50 μm.</p> <p><b>(F)</b> The <i>exo70B1-3</i> mutants showed enhanced MAP kinase activation upon <i>G. cichoracearum</i> infection. Four-week-old plants were infected with <i>G. cichoracearum</i> at high inoculum densities. The immunoblot analysis was performed using anti-pTEpY antibody to examine MAPK activation, and anti-MPK3 or anti-MPK6 to show accumulation of MPK3 or MPK6 protein, respectively. Individual MPKs are indicated by arrows. Ponceau S staining of RuBisCO is shown as loading control.</p></div