Arabidopsis membrane protein AMAR1 interaction with type III effector XopAM triggers a hypersensitive response.

The efficient infection of plants by the bacteria Xanthomonas campestris pv. campestris (Xcc) depends on its type III effectors (T3Es). Although the functions of AvrE family T3Es have been reported in some bacteria, the member XopAM in Xcc has not been studied. As XopAM has low sequence similarity to reported AvrE-T3Es and different reports have shown that these T3Es have different targets in hosts, we investigated the functions of XopAM in the Xcc-plant interaction. Deletion of xopAM from Xcc reduced its virulence in cruciferous crops but increased virulence in Arabidopsis (Arabidopsis thaliana) Col-0, indicating that XopAM may perform opposite functions depending on the host species. We further found that XopAM is a lipase that may target the cytomembrane and that this activity might be enhanced by its membrane-targeted protein XOPAM-ACTIVATED RESISTANCE 1 (AMAR1) in Arabidopsis Col-0. The binding of XopAM to AMAR1 induced an intense hypersensitive response that restricted Xcc proliferation. Our results showed that the roles of XopAM in Xcc infection are not the same as those of other AvrE-T3Es, indicating that the functions of this type of T3E have differentiated during long-term bacterium‒host interactions.

Roles of CgEde1 and CgMca in Development and Virulence of Colletotrichum gloeosporioides.

Anthracnose, induced by Colletotrichum gloeosporioides, poses a substantial economic threat to rubber tree yields and various other tropical crops. Ede1, an endocytic scaffolding protein, plays a crucial role in endocytic site initiation and maturation in yeast. Metacaspases, sharing structural similarities with caspase family proteases, are essential for maintaining cell fitness. To enhance our understanding of the growth and virulence of C. gloeosporioides, we identified a homologue of Ede1 (CgEde1) in C. gloeosporioides. The knockout of CgEde1 led to impairments in vegetative growth, conidiation, and pathogenicity. Furthermore, we characterized a weakly interacted partner of CgEde1 and CgMca (orthologue of metacaspase). Notably, both the single mutant ΔCgMca and the double mutant ΔCgEde1/ΔCgMca exhibited severe defects in conidiation and germination. Polarity establishment and pathogenicity were also disrupted in these mutants. Moreover, a significantly insoluble protein accumulation was observed in ΔCgMca and ΔCgEde1/ΔCgMca strains. These findings elucidate the mechanism by which CgEde1 and CgMca regulates the growth and pathogenicity of C. gloeosporioides. Their regulation involves influencing conidiation, polarity establishment, and maintaining cell fitness, providing valuable insights into the intricate interplay between CgEde1 and CgMca in C. gloeosporioides.

CgCFEM1 Is Required for the Full Virulence of Colletotrichum gloeosporioides.

Colletotrichum gloeosporioides is widely distributed and causes anthracnose on many crops, resulting in serious economic losses. Common fungal extracellular membrane (CFEM) domain proteins have been implicated in virulence and their interaction with the host plant, but their roles in C. gloeosporioides are still unknown. In this study, a CFEM-containing protein of C. gloeosporioides was identified and named as CgCFEM1. The expression levels of CgCFEM1 were found to be markedly higher in appressoria, and this elevated expression was particularly pronounced during the initial stages of infection in the rubber tree. Absence of CgCFEM1 resulted in impaired pathogenicity, accompanied by notable perturbations in spore morphogenesis, conidiation, appressorium development and primary invasion. During the process of appressorium development, the absence of CgCFEM1 enhanced the mitotic activity in both conidia and germ tubes, as well as compromised conidia autophagy. Rapamycin was found to basically restore the appressorium formation, and the activity of target of rapamycin (TOR) kinase was significantly induced in the CgCFEM1 knockout mutant (∆CgCFEM1). Furthermore, CgCFEM1 was proved to suppress chitin-triggered reactive oxygen species (ROS) accumulation and change the expression patterns of defense-related genes. Collectively, we identified a fungal effector CgCFEM1 that contributed to pathogenicity by regulating TOR-mediated conidia and appressorium morphogenesis of C. gloeosporioides and inhibiting the defense responses of the rubber tree.

Histone acetyltransferase HAM1 interacts with molecular chaperone DNAJA2 and confers immune responses through salicylic acid biosynthetic genes in cassava.

Cassava bacterial blight (CBB) is one of the most serious diseases in cassava production, so it is essential to explore the underlying mechanism of immune responses. Histone acetylation is an important epigenetic modification, however, its relationship with cassava disease resistance remains unclear. Here, we identified 10 histone acetyltransferases in cassava and found that the transcript of MeHAM1 showed the highest induction to CBB. Functional analysis showed that MeHAM1 positively regulated disease resistance to CBB through modulation of salicylic acid (SA) accumulation. Further investigation revealed that MeHAM1 directly activated SA biosynthetic genes' expression via promoting lysine 9 of histone 3 (H3K9) acetylation and lysine 5 of histone 4 (H4K5) acetylation of these genes. In addition, molecular chaperone MeDNAJA2 physically interacted with MeHAM1, and MeDNAJA2 also regulated plant immune responses and SA biosynthetic genes. In conclusion, this study illustrates that MeHAM1 and MeDNAJA2 confer immune responses through transcriptional programming of SA biosynthetic genes via histone acetylation. The MeHAM1 & MeDNAJA2-SA biosynthesis module not only constructs the direct relationship between histone acetylation and cassava disease resistance, but also provides gene network with potential value for genetic improvement of cassava disease resistance.

Histone deacetylase 9 regulates disease resistance through fine-tuning histone deacetylation of melatonin biosynthetic genes and melatonin accumulation in cassava.

Melatonin participates in plant growth and development and biotic and abiotic stress responses. Histone acetylation regulates many plant biological processes via transcriptional reprogramming. However, the direct relationship between melatonin and histone acetylation in plant disease resistance remains unclear. In this study, we identified cassava bacterial blight (CBB) responsive histone deacetylase 9 (HDA9), which negatively regulated disease resistance to CBB by reducing melatonin content. In addition, exogenous melatonin alleviated disease sensitivity of MeHDA9 overexpressed plants to CBB. Importantly, MeHDA9 inhibited the expression of melatonin biosynthetic genes through decreasing lysine 5 of histone 4 (H4K5) acetylation at the promoter regions of melatonin biosynthetic genes, thereby modulating melatonin accumulation in cassava. Furthermore, protein phosphatase 2C 12 (MePP2C12) interacted with MeHDA9 in vivo and in vitro, and it was involved in MeHDA9-mediated disease resistance via melatonin biosynthetic pathway. In summary, this study highlights the direct interaction between histone deacetylation and melatonin biosynthetic genes in cassava disease resistance via histone deacetylation, providing new insights into the genetic improvement of disease resistance via epigenetic regulation of melatonin level in tropical crops.

CgMFS1, a Major Facilitator Superfamily Transporter, Is Required for Sugar Transport, Oxidative Stress Resistance, and Pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis.

Colletotrichum gloeosporioides is the main causal agent of anthracnose in various plant species. Determining the molecular mechanisms underlying the pathogenicity and fungicide resistance of C. gloeosporioides could help build new strategies for disease control. The major facilitator superfamily (MFS) has multiple roles in the transport of a diverse range of substrates. In the present study, an MFS protein CgMFS1 was characterized in C. gloeosporioides. This protein contains seven transmembrane domains, and its predicted 3D structure is highly similar to the reported hexose transporters. To investigate the biological functions of CgMFS1, the gene knock-out mutant ΔCgMFS1 was constructed. A colony growth assay showed that the mutant was remarkably decreased in vegetative growth in minimal medium supplemented with monosaccharides and oligosaccharides as the sole carbon sources, whereas it showed a similar growth rate and colony morphology as wild types when using soluble starch as the carbon source. A stress assay revealed that CgMFS1 is involved in oxidative stress but not in the fungicide resistance of C. gloeosporioides. Furthermore, its pathogenicity was significantly impaired in the mutant, although its appressorium formation was not affected. Our results demonstrate that CgMFS1 is required for sugar transport, resistance to oxidative stress, and the pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis.

The dual interplay of RAV5 in activating nitrate reductases and repressing catalase activity to improve disease resistance in cassava.

Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) seriously affects cassava yield. Nitrate reductase (NR) plays an important role in plant nitrogen metabolism in plants. However, the in vivo role of NR and the corresponding signalling pathway remain unclear in cassava. In this study, we isolated MeNR1/2 and revealed their novel upstream transcription factor MeRAV5. We also identified MeCatalase1 (MeCAT1) as the interacting protein of MeRAV5. In addition, we investigated the role of MeCatalase1 and MeRAV5-MeNR1/2 module in cassava defence response. MeNRs positively regulates cassava disease resistance against CBB through modulation of nitric oxide (NO) and extensive transcriptional reprogramming especially in mitogen-activated protein kinase (MAPK) signalling. Notably, MeRAV5 positively regulates cassava disease resistance through the coordination of NO and hydrogen peroxide (H2 O2 ) level. On the one hand, MeRAV5 directly activates the transcripts of MeNRs and NO level by binding to CAACA motif in the promoters of MeNRs. On the other hand, MeRAV5 interacts with MeCAT1 to inhibit its activity, so as to negatively regulate endogenous H2 O2 level. This study highlights the precise coordination of NR activity and CAT activity by MeRAV5 through directly activating MeNRs and interacting with MeCAT1 in plant immunity.

Computational determination of gene age and characterization of evolutionary dynamics in human.

Genes originate at different evolutionary time scales and possess different ages, accordingly presenting diverse functional characteristics and reflecting distinct adaptive evolutionary innovations. In the past decades, progresses have been made in gene age identification by a variety of methods that are principally based on comparative genomics. Here we summarize methods for computational determination of gene age and evaluate the effectiveness of different computational methods for age identification. Our results show that improved age determination can be achieved by combining homolog clustering with phylogeny inference, which enables more accurate age identification in human genes. Accordingly, we characterize evolutionary dynamics of human genes based on an extremely long evolutionary time scale spanning ~4,000 million years from archaea/bacteria to human, revealing that young genes are clustered on certain chromosomes and that Mendelian disease genes (including monogenic disease and polygenic disease genes) and cancer genes exhibit divergent evolutionary origins. Taken together, deciphering genes' ages as well as their evolutionary dynamics is of fundamental significance in unveiling the underlying mechanisms during evolution and better understanding how young or new genes become indispensable integrants coupled with novel phenotypes and biological diversity.

Label free proteomics and systematic analysis of secretome reveals effector candidates regulated by SGE1 and FTF1 in the plant pathogen Fusarium oxysporum f. sp. cubense tropical race 4.

BACKGROUND: Phytopathogens secreted effectors during host colonization to suppress or trigger plant immunity. Identification of new effectors is one of the research focuses in recent years. There is only a limited knowledge about effectors of Fusarium oxysporum f. sp. Cubense tropical race 4 (Foc TR4), the causal agent of wilt disease in Cavendish banana. RESULTS: Two transcription factors, SGE1 and FTF1, were constitutively over-expressed in Foc TR4 to partially mimic the in-planta state. Secreted proteins with high purity were prepared through a two-round extraction method. Then the secretome were analyzed via label free proteomics method. A total of 919 non-redundant proteins were detected, of which 74 proteins were predicted to be effector candidates. Among these candidates, 29 were up-regulated and 13 down-regulated in the strain over-expressing SGE1 and FTF1, 8 were up-regulated and 4 down-regulated in either SGE1 or FTF1 over expression strain. CONCLUSIONS: Through label free proteomics analysis, a series of effector candidates were identified in secretome of Foc TR4. Our work put a foundation for functional research of these effectors.

HbWRKY40 plays an important role in the regulation of pathogen resistance in Hevea brasiliensis.

KEY MESSAGE: Overexpression of HbWRKY40 induces ROS burst in tobacco and increases disease resistance in Arabidopsis; RNA-seq and ChIP assays revealed the regulatory network of HbWRKY40 in plant defense. WRKY, a family of plant transcription factors, are involved in the regulation of numerous biological processes. In rubber tree Hevea brasiliensis, the roles of WRKYs remain poorly understood. In the present study, a total of 111 genes encoding putative HbWRKY proteins were identified in the H. brasiliensis genome. Among these genes, HbWRKY40 transcripts were significantly induced by Colletotrichum gloeosporioides and salicylic acid. To assess its roles in plant defense, HbWRKY40 was over-expressed in Nicotiana benthamiana and Arabidopsis thaliana. The results showed that HbWRKY40 significantly induced reactive oxygen species burst in N. benthamiana and increased resistance of Arabidopsis against Botrytis cinerea. Transient expression in mesophyll cell protoplasts of H. brasiliensis showed that HbWRKY40 localizes at nuclei. In addition, transcripts of 145 genes were significantly up-regulated and 6 genes were down-regulated in the protoplasts over-expressing HbWRKY40 based on the RNA-seq analysis. Among these potential downstream targets, 12 genes contain potential WRKY-binding sites at the promoter regions. Further analysis through chromatin immunoprecipitation revealed that 10 of these 12 genes were the downstream targets of HbWRKY40. Taken together, our findings indicate that HbWRKY40 plays an important role in the disease resistance by regulating defense-associated genes in H. brasiliensis.

Selection of Optimized Reference Genes for qRT-PCR Normalization in Xanthomonas campestris pv. campestris Cultured in Different Media.

Black rot is a cruciferous disease caused by Xanthomonas campestris pv. campestris (Xcc) and results in significant economic losses worldwide; therefore, elucidation of the mechanism of Xcc pathogenesis is urgently required. In this study, we aimed to select optimized reference genes to verify the relative quantification of virulent genes in Xcc. Xcc strains were cultured in three different media [basic medium (MMX), hrp-inducing medium (MMXC) and rich medium (NYG)] and the expression stability of five candidate genes [thymidylate synthase (thyA), DNA gyrase subunit B (gyrB), DNA-directed RNA polymerase subunit beta, glyceraldehyde-3-phosphate dehydrogenase and 16S ribosomal RNA (16S rRNA)] was evaluated using BestKeeper, GeNorm, and NormFinder software programs. Quantitative real-time PCR (qRT-PCR) analysis confirmed that two Xcc effector genes were hrpX/hrpG-regulated in MMXC using selected genes as controls. Finally, gyrB and thyA were validated as the optimized reference genes of Xcc cultured in MMXC, and qRT-PCR analysis was demonstrated to be an efficient alternative to Gus-activity detection for the analysis of Xcc expression. This information will be useful in the future studies of Xcc, especially those seeking new functional genes.

AtKATANIN1 Modulates Microtubule Depolymerization and Reorganization in Response to Salt Stress in Arabidopsis.

The microtubule cytoskeleton is a dynamic system that plays vital roles in fundamental cellular processes and in responses to environmental stumili. Salt stress induced depolymerization and reorganization of microtubules are believed to function in the promotion of survival in Arabidopsis. Microtubule-severing enzyme ATKATANIN1 (AtKTN1) is recognized as a MAP that help to maintain organized microtubule structure. To date, whether AtKTN1 is involved in response to salt stress in Arabidopsis remains unknown. Here, our phenotypic analysis showed that the overexpression of AtKTN1 decreased tolerance to salt stress, whereas the knock-out of AtKTN1 increased salt tolerance in the early stage but decreased salt tolerance in the later stage. Microscopic analysis revealed that microtubule organization and dynamics are distorted in both overexpression and mutant cells which, in turn, resulted in an abnormal disassembly and reorganization under salt stress. Moreover, qRT analysis revealed that stress-responsive genes were down-regulated in overexpression and mutant cells compared to WT cells under salt stress. Taken together, our results indicated roles of AtKTN1 in modulating microtubule organization, salt-stress induced microtubule disruption and recovery, and its involvement in stress-related signaling pathways.

Differential Function of Endogenous and Exogenous Abscisic Acid during Bacterial Pattern-Induced Production of Reactive Oxygen Species in Arabidopsis.

Abscisic acid (ABA) plays important roles in positively or negatively regulating plant disease resistance to pathogens. Here, we reassess the role of endogenous and exogenous ABA by using: 35S::ABA2, a previously reported transgenic Arabidopsis line with increased endogenous ABA levels; aba2-1, a previously reported ABA2 mutant with reduced endogenous ABA levels; and exogenous application of ABA. We found that bacterial susceptibility promoted by exogenous ABA was suppressed in 35S::ABA2 plants. The 35S::ABA2 and aba2-1 plants displayed elevated and reduced levels, respectively, of bacterial flagellin peptide (flg22)-induced H2O2. Surprisingly, ABA pre-treatment reduced flg22-induced H2O2 generation. Exogenous, but not endogenous ABA, increased catalase activity. Loss of nicotinamide adenine dinucleotide phosphate oxidase genes, RBOHD and RBOHF, restored exogenous ABA-promoted bacterial susceptibility of 35S::ABA2 transgenic plants. In addition, endogenous and exogenous ABA had similar effects on callose deposition and salicylic acid (SA) signaling. These results reveal an underlying difference between endogenous and exogenous ABA in regulating plant defense responses. Given that some plant pathogens are able to synthesize ABA and affect endogenous ABA levels in plants, our results highlight the importance of reactive oxygen species in the dual function of ABA during plant-pathogen interactions.

The YebN Leader RNA Acts as a Mn2+ Sensor Required for the Interaction of Xanthomonas oryzae and Rice.

Riboswitches are RNA elements that sense metabolites and control gene expression. Recently, the yybP-ykoY riboswitches were found to sense manganese (Mn2+) and regulate the expression of diverse genes. Here, we show that the leader RNA (a yybP-ykoY RNA) of yebN in Xanthomonas oryzae pv. oryzae also functions as a sensor of Mn2+. This leader RNA detects Mn2+ levels in plants and is essential to X. oryzae pv. oryzae virulence. Our data also indicate that Mn2+ is not only required as a microelement for plant growth but also acts as a defense molecule to inhibit pathogen growth. This finding highlights that Mn2+ plays important roles in pathogen-plant interactions and that the yebN leader RNA can be a target candidate for anti-X. oryzae pv. oryzae drug development.

Molecular identification of GAPDHs in cassava highlights the antagonism of MeGAPCs and MeATG8s in plant disease resistance against cassava bacterial blight.

KEY MESSAGE: MeGAPCs were identified as negative regulators of plant disease resistance, and the interaction of MeGAPCs and MeATG8s was highlighted in plant defense response. As an important enzyme of glycolysis metabolic pathway, glyceraldehyde-3-P dehydrogenase (GAPDH) plays important roles in plant development, abiotic stress and immune responses. Cassava (Manihot esculenta) is most important tropical crop and one of the major food crops, however, no information is available about GAPDH gene family in cassava. In this study, 14 MeGAPDHs including 6 cytosol GAPDHs (MeGAPCs) were identified from cassava, and the transcripts of 14 MeGAPDHs in response to Xanthomonas axonopodis pv manihotis (Xam) indicated their possible involvement in immune responses. Further investigation showed that MeGAPCs are negative regulators of disease resistance against Xam. Through transient expression in Nicotiana benthamiana, we found that overexpression of MeGAPCs led to decreased disease resistance against Xam. On the contrary, MeGAPCs-silenced cassava plants through virus-induced gene silencing (VIGS) conferred improved disease resistance. Notably, MeGAPCs physically interacted with autophagy-related protein 8b (MeATG8b) and MeATG8e and inhibited autophagic activity. Moreover, MeATG8b and MeATG8e negatively regulated the activities of NAD-dependent MeGAPDHs, and are involved in MeGAPCs-mediated disease resistance. Taken together, this study highlights the involvement of MeGAPCs in plant disease resistance, through interacting with MeATG8b and MeATG8e.

RAV transcription factors are essential for disease resistance against cassava bacterial blight via activation of melatonin biosynthesis genes.

With 1 AP2 domain and 1 B3 domain, 7 MeRAVs in apetala2/ethylene response factor (AP2/ERF) gene family have been identified in cassava. However, the in vivo roles of these remain unknown. Gene expression assays showed that the transcripts of MeRAVs were commonly regulated after Xanthomonas axonopodis pv manihotis (Xam) and MeRAVs were specifically located in plant cell nuclei. Through virus-induced gene silencing (VIGS) in cassava, we found that MeRAV1 and MeRAV2 are essential for plant disease resistance against cassava bacterial blight, as shown by the bacterial propagation of Xam in plant leaves. Through VIGS in cassava leaves and overexpression in cassava leave protoplasts, we found that MeRAV1 and MeRAV2 positively regulated melatonin biosynthesis genes and the endogenous melatonin level. Further investigation showed that MeRAV1 and MeRAV2 are direct transcriptional activators of 3 melatonin biosynthesis genes in cassava, as evidenced by chromatin immunoprecipitation-PCR in cassava leaf protoplasts and electrophoretic mobility shift assay. Moreover, cassava melatonin biosynthesis genes also positively regulated plant disease resistance. Taken together, this study identified MeRAV1 and MeRAV2 as common and upstream transcription factors of melatonin synthesis genes in cassava and revealed a model of MeRAV1 and MeRAV2-melatonin biosynthesis genes-melatonin level in plant disease resistance against cassava bacterial blight.

Melatonin biosynthesis enzymes recruit WRKY transcription factors to regulate melatonin accumulation and transcriptional activity on W-box in cassava.

Melatonin is widely involved in growth, development, and stress responses in plants. Although the melatonin synthesis enzymes have been identified in various plants, their interacting proteins remain unknown. Herein, overexpression of tryptophan decarboxylase 2 (MeTDC2)-interacting proteins, N-acetylserotonin O-methyltransferase 2 (MeASMT2) interacting proteins, and N-acetylserotonin O-methyltransferase 3 (MeASMT3) in cassava leaf protoplasts resulted in more melatonin than when other enzymes were overexpressed. Through yeast two-hybrid, 14 MeTDC2-interacting proteins, 24 MeASMT2 interacting proteins, and 9 MeASMT3-interacting proteins were identified. Notably, we highlighted MeWRKY20 and MeWRKY75 as common interacting proteins of the 3 enzymes, as evidenced by yeast two-hybrid, and in vivo bimolecular fluorescence complementation (BiFC). Moreover, co-overexpression of MeTDC2/MeASMT2/3 with MeWRKY20/75 in cassava leaf protoplasts did not only activated the transcriptional activities of MeWRKY20 and MeWRKY75 on W-box, but also induced the effects of MeTDC2, MeASMT2/3 on endogenous melatonin levels. Taken together, 3 melatonin synthesis enzymes (MeTDC2, MeASMT2/3) interact with MeWRKY20/75 to form a protein complex in cassava. This information significantly extends the knowledge of the complex modulation of plant melatonin signaling.

A Xanthomonas uridine 5'-monophosphate transferase inhibits plant immune kinases.

Plant innate immunity is activated on the detection of pathogen-associated molecular patterns (PAMPs) at the cell surface, or of pathogen effector proteins inside the plant cell. Together, PAMP-triggered immunity and effector-triggered immunity constitute powerful defences against various phytopathogens. Pathogenic bacteria inject a variety of effector proteins into the host cell to assist infection or propagation. A number of effector proteins have been shown to inhibit plant immunity, but the biochemical basis remains unknown for the vast majority of these effectors. Here we show that the Xanthomonas campestris pathovar campestris type III effector AvrAC enhances virulence and inhibits plant immunity by specifically targeting Arabidopsis BIK1 and RIPK, two receptor-like cytoplasmic kinases known to mediate immune signalling. AvrAC is a uridylyl transferase that adds uridine 5'-monophosphate to and conceals conserved phosphorylation sites in the activation loop of BIK1 and RIPK, reducing their kinase activity and consequently inhibiting downstream signalling.