Xanthomonas campestris pv. musacearum bacterial infection induces organ-specific callose and hydrogen peroxide production in banana

Xanthomonas campestris pv. musacearum (Xcm) bacteria cause banana Xanthomonas wilt (BXW), the most destructive disease of bananas in East and Central Africa. During early stages of infection in susceptible banana cultivars, incomplete systemic movement of Xcm limits bacterial colonization in the upper organs. Mechanistic basis of this delayed movement is unknown. We hypothesized that Xcm infection triggers basal pattern triggered immune (PTI) responses whose spatial and temporal variability along banana’s anatomical structure accounts for initially limiting Xcm in upper organs. Hence, we examined PTI responses such as callose deposition and hydrogen peroxide (H2O2) production in different organs in response to Xcm infection in BXW susceptible Kayinja and Mbwazirume banana cultivars and wild resistant progenitor Musa balbisiana. Xcm-induced callose increased and peaked at 14 days post inoculation (dpi) and 28dpi as assessed by fluorescence microscopy and enzyme-linked immunosorbent assays, respectively. The levels of Xcm-induced H2O2 and callose were highest in the pseudostems and corms, respectively, and were independent of host susceptibility or resistance to BXW. H2O2 production showed a biphasic transient pattern with an initial increase at 1-hour post Xcm-inoculation (hpi), followed by a decline 3-6hpi and then a second increase by 12hpi. Our findings point to organ-specific responses to Xcm infection in bananas. The corm which doubles as a subterranean parenating organ and interface between mother plants and lateral shoots, was the most responsive organ in callose production while the pseudostem was the most responsive organ in H2O2 production, suggesting the significance of these organs in banana response to BXW.

The E3 Ubiquitin Ligase ATL9 Affects Expression of Defense Related Genes, Cell Death and Callose Deposition in Response to Fungal Infection

Plants use diverse strategies to defend themselves from biotic stresses in nature, which include the activation of defense gene expression and a variety of signal transduction pathways. Previous studies have shown that protein ubiquitination plays a critical role in plant defense responses, however the details of its function remain unclear. Our previous work has shown that increasing expression levels of ATL9, an E3 ubiquitin ligase in Arabidopsis thaliana, increased resistance to infection by the fungal pathogen, Golovinomyces cichoracearum. In this study, we demonstrate that the defense-related proteins PDF1.2, PCC1 and FBS1 directly interact with ATL9 and are targeted for degradation to the proteasome by ATL9. The expression levels of PDF1.2, PCC1 and FBS1 are decreased in T-DNA insertional mutants of atl9 and T-DNA insertional mutants of pdf1.2, pcc1 and fbs1 are more susceptible to fungal infection. In addition, callose is more heavily deposited at infection sites in the mutants of atl9, fbs1, pcc1 and pdf1.2. Overexpression of ATL9 and of mutants in fbs1, pcc1 and pdf1.2 showed increased levels of cell death during infection. Together these results indicate that ubiquitination, cell death and callose deposition may work together to enhance defense responses to fungal pathogens.

Pseudomonas syringae pv. actinidiae Effector HopAU1 Interacts with Calcium-Sensing Receptor to Activate Plant Immunity

Kiwifruit canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a destructive pathogen that globally threatens the kiwifruit industry. Understanding the molecular mechanism of plant-pathogen interaction can accelerate applying resistance breeding and controlling plant diseases. All known effectors secreted by pathogens play an important role in plant-pathogen interaction. However, the effectors in Psa and their function mechanism remain largely unclear. Here, we successfully identified a T3SS effector HopAU1 which had no virulence contribution to Psa, but could, however, induce cell death and activate a series of immune responses by agroinfiltration in Nicotiana benthamiana, including elevated transcripts of immune-related genes, accumulation of reactive oxygen species (ROS), and callose deposition. We found that HopAU1 interacted with a calcium sensing receptor in N. benthamiana (NbCaS) as well as its close homologue in kiwifruit (AcCaS). More importantly, silencing CaS by RNAi in N. benthamiana greatly attenuated HopAU1-triggered cell death, suggesting CaS is a crucial component for HopAU1 detection. Further researches showed that overexpression of NbCaS in N. benthamiana significantly enhanced plant resistance against Sclerotinia sclerotiorum and Phytophthora capsici, indicating that CaS serves as a promising resistance-related gene for disease resistance breeding. We concluded that HopAU1 is an immune elicitor that targets CaS to trigger plant immunity.

‘Candidatus Liberibacter asiaticus’-Encoded BCP Peroxiredoxin Suppresses Lipopolysaccharide-Mediated Defense Signaling and Nitrosative Stress in Planta

The lipopolysaccharides (LPS) of Gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic ‘Candidatus Liberibacter’ spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid (VLCFA) modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on non-host tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host citrus (Citrus sinensis, confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione (GSH) pool, callose deposition and activation of the salicylic acid (SA) and azelaic acid (AzA) signaling networks. Transient expression of ‘Ca. L. asiaticus’ BCP peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that ‘Ca. L. asiaticus’ BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. ‘Ca. L. asiaticus’ BCP peroxiredoxin (a) attenuates NO-mediated SAR signaling and (b) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of ‘Ca. L. asiaticus’ acquisition and transmission by fecund psyllids throughout the limited flush period in citrus.

Fungal oxysterol-binding protein-related proteins promote pathogen virulence and activate plant immunity

Oxysterol-binding protein-related proteins (ORPs) are a conserved class of lipid transfer proteins that are closely involved in multiple cellular processes in eukaryotes but their roles in plant-pathogen interactions are mostly unknown. We showed that transient expression of ORPs of Magnaporthe oryzae (MoORPs) in Nicotiana benthamina plants triggered oxidative burst and cell death; treatment of tobacco Bright Yellow-2 suspension cells with recombinant MoORPs elicited the production of reactive oxygen species. Despite that ORPs are normally described as intracellular proteins, we detected MoORPs in fungal cultural filtrates and intercellular fluids from barley plants infected with the fungus. More importantly, infiltration of Arabidopsis plants with recombinant Arabidopsis or fungal ORPs activated oxidative burst, callose deposition, PR1 gene expression, and enhanced plant disease resistance, implying that ORPs may function as endogenous and exogenous danger signals triggering plant innate immunity. Extracellular application of fungal ORPs exerted an opposite impact on salicylic acid and jasmonic acid/ethylene signaling pathways. The Brassinosteroid Insensitive 1-associated Kinase 1 was dispensable for the ORP-activated defense. Besides, simultaneous knockout of MoORP1 and MoORP3 abolished fungal colony radial growth and conidiation, whereas double knockout of MoORP1 and MoORP2 compromised fungal virulence on barley and rice plants. These observations collectively highlight the multifaceted role of MoORPs in the modulation of plant innate immunity and promotion of fungal development and virulence in M. oryzae.

Arabidopsis Spliceosome Factor SmD3 Modulates Immunity to Pseudomonas syringae Infection

SmD3 is a core component of the small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. The role of Arabidopsis SmD3 in plant immunity was assessed by testing sensitivity of smd3a and smd3b mutants to Pseudomonas syringae pv. tomato (Pst) DC3000 infection and its pathogenesis effectors flagellin (flg22), EF-Tu (elf18) and coronatine (COR). Both smd3 mutants exhibited enhanced susceptibility to Pst accompanied by marked changes in the expression of key pathogenesis markers. mRNA levels of major biotic stress response factors were also altered upon treatment with Pseudomonas effectors. Our genome-wide transcriptome analysis of the smd3b-1 mutant infected with Pst, verified by northern and RT-qPCR, showed that lack of SmD3-b protein deregulates defense against Pst infection at the transcriptional and posttranscriptional levels including defects in splicing and an altered pattern of alternative splicing. Importantly, we show that SmD3-b dysfunction impairs mainly stomatal immunity as a result of defects in stomatal development. We propose that it is the malfunction of the stomata that is the primary cause of an altered mutant response to the pathogen. Other changes in the smd3b-1 mutant involved enhanced elf18- and flg22-induced callose deposition, reduction of flg22-triggered production of early ROS and boost of secondary ROS caused by Pst infection. Together, our data indicate that SmD3 contributes to the plant immune response possibly via regulation of mRNA splicing of key pathogenesis factors.

Silencing an E3 Ubiquitin Ligase Gene OsJMJ715 Enhances the Resistance of Rice to a Piercing-Sucking Herbivore by Activating ABA and JA Signaling Pathways

The RING-type E3 ubiquitin ligases play an important role in plant growth, development, and defense responses to abiotic stresses and pathogens. However, their roles in the resistance of plants to herbivorous insects remain largely unknown. In this study, we isolated the rice gene OsJMJ715, which encodes a RING-domain containing protein, and investigated its role in rice resistance to brown planthopper (BPH, Nilaparvata lugens). OsJMJ715 is a nucleus-localized E3 ligase whose mRNA levels were upregulated by the infestation of gravid BPH females, mechanical wounding, and treatment with JA or ABA. Silencing OsJMJ715 enhanced BPH-elicited levels of ABA, JA, and JA-Ile as well as the amount of callose deposition in plants, which in turn increased the resistance of rice to BPH by reducing the feeding of BPH and the hatching rate of BPH eggs. These findings suggest that OsJMJ715 negative regulates the BPH-induced biosynthesis of ABA, JA, and JA-Ile and that BPH benefits by enhancing the expression of OsJMJ715.

Genome-wide analysis of the CalS gene family in cotton reveals their potential roles in fiber development and responses to stress

Callose deposition occurs during plant growth and development, as well as when plants are under biotic and abiotic stress. Callose synthase is a key enzyme for the synthesis of callose. In this study, 27, 28, 16, and 15 callose synthase family members were identified in Gossypium hirsutum, Gossypium barbadense, Gossypium raimondii, and Gossypium arboreum using the sequence of Arabidopsis callose synthase. The CalSs were divided into five groups by phylogenetic, gene structure, and conservative motif analysis. The conserved motifs and gene structures of CalSs in each group were highly similar. Based on the analysis of cis-acting elements, it is inferred that GhCalSs were regulated by abiotic stress. WGD/Segmental duplication promoted the amplification of the CalS gene in cotton, and purification selection had an important function in the CalS family. The transcriptome data and qRT-PCR under cold, heat, salt, and PEG treatments showed that GhCalSs were involved in abiotic stress. The expression patterns of GhCalSs were different in various tissues. We predicted that GhCalS4, which was highly expressed in fibers, had an important effect on fiber elongation. Hence, these results help us understand the role of GhCalSs in fiber development and stress response.

Biochemical and Cytological Interactions Between Callose Synthase and Microtubules in the Tobacco Pollen Tube

Callose is a cell wall polysaccharide involved in several fundamental biological processes, ranging from plant development to response to abiotic and biotic stresses. To understand how callose deposition is regulated, it is important to know how its synthesizing enzyme, i.e., callose synthase, is regulated and if it interacts with vesicular-cytoskeletal system of plant cells. Actin filaments are thought to determine the long-range distribution of callose synthase through transport vesicles. Unlike other enzymes (such as cellulose synthase) that synthesize cell wall polysaccharides, the spatial and biochemical relationships between callose synthase and microtubules are poorly understood. Some experimental evidence already support the association between callose synthase and tubulin, however, despite its importance in maintaining plant integrity, knowledge about regulation of callose biosynthesis is still limited. Here we investigated the association between callose synthase and cytoskeleton by biochemical and ultrastructural analyses in a model system, pollen tube, where callose is an essential cell wall component. Native 2-D electrophoresis and isolation of the callose synthase complex confirmed that callose synthase is associated with tubulin and can interface with cortical microtubules. In contrast, actin and sucrose synthase (which supplies UDP-glucose to callose synthase) are not permanently associated with callose synthase. Immunogold labeling showed strong colocalization of the enzyme and microtubules; this association is occasionally mediated by vesicles. The association between callose synthase and vesicles was also demonstrated by co-distribution between the enzyme and Rab11b; in addition, the not homogeneous distribution of callose synthase in cell membranes is also shown by analysis of membrane microdomains.