Role of a xyloglucan-specific endo-beta-1,4-glucanase inhibitor in the interactions of Nicotiana benthamiana with Colletotrichum destructivum, C. orbiculare or Pseudomonas syringae pv. tabaci

A xyloglucan-specific endo-β-1,4-glucanase inhibitor cDNA, NbXEGIP1 , was amplified from diseased leaves of Nicotiana benthamiana . The sequence was similar to the tomato xyloglucan-specific endo-β-1,4-glucanase inhibitor (XEGIP) and tobacco nectarin IV genes that have been described as binding and inactivating fungal Family 12 xyloglucan-specific endo-β-1,4-glucanases. Expression of NbXEGIP1 was not detected in healthy leaves, but the gene was induced during the later stages of infection by the fungi Colletotrichum destructivum and C. orbiculare . Induction of NbXEGIP1 also occurred during disease development by the bacterium Pseudomonas syringae pv. tabaci and during the hypersensitive response produced by P. syringae pv. tabaci expressing avrPto . A portion of NbXEGIP1 was cloned into a tobacco rattle virus vector for virus-induced gene silencing in N. benthamiana . Silencing NbXEGIP1 did not affect the interactions with either Colletotrichum species but did significantly reduce population levels of P. syringae pv. tabaci in the compatible interaction and P. syringae pv. tabaci expressing avrPto in the incompatible interaction. In the susceptible response to P. syringae pv. tabaci , silencing of NbXEGIP1 also resulted in visibly wilted leaves several hours prior to necrosis, which was not observed in control plants. This was related to a significantly higher level of electrolyte leakage and higher expression of a defensin gene from infected NbXEGIP1 -silenced leaves compared with control leaves. Silencing appeared to be specific as it did not affect expression of a related gene, NbXEGIP2 . NbXEGIP1 may act as an inhibitor of a bacterial enzyme that degrades the xyloglucan–cellulose plant cell-wall network, and degradation of the cell wall results in host membrane disruption and signalling of defence responses.     

Cell death of Nicotiana benthamiana is induced by secreted protein NIS1 of colletotrichum orbiculare and is suppressed by a homologue of CgDN3

Colletotrichum orbiculare, the causal agent of cucumber anthracnose, infects Nicotiana benthamiana. Functional screening of C. orbiculare cDNAs in a virus vector-based plant expression system identified a novel secreted protein gene, NIS1, whose product induces cell death in N. benthamiana. Putative homologues of NIS1 are present in selected members of fungi belonging to class Sordariomycetes, Dothideomycetes, or Orbiliomycetes. Green fluorescent protein-based expression studies suggested that NIS1 is preferentially expressed in biotrophic invasive hyphae. NIS1 lacking signal peptide did not induce NIS1-triggered cell death (NCD), suggesting apoplastic recognition of NIS1. NCD was prevented by virus-induced gene silencing of SGT1 and HSP90, indicating the dependency of NCD on SGT1 and HSP90. Deletion of NIS1 had little effect on the virulence of C. orbiculare against N. benthamiana, suggesting possible suppression of NCD by C. orbiculare at the postinvasive stage. The CgDN3 gene of C. gloeosporioides was previously identified as a secreted protein gene involved in suppression of hypersensitive-like response in Stylosanthes guianensis. Notably, we found that NCD was suppressed by the expression of a CgDN3 homologue of C. orbiculare. Our findings indicate that C. orbiculare expresses NIS1 at the postinvasive stage and suggest that NCD could be repressed via other effectors, including the CgDN3 homologue.     

Use of green fluorescent protein to quantify the growth of Colletotrichum during infection of tobacco

To develop a quantitative assay of fungal growth inside plant tissues, strains of Colletotrichum destructivum and Colletotrichum orbiculare were transformed with a modified green fluorescent protein (GFP) gene fused with a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus nidulans. Transformants expressed GFP in culture and had the same growth rate and general appearance as the wild type. GFP was observed in all fungal structures during infection of leaves of Nicotiana benthamiana, except for the melanized appressoria and setae. The timing and appearance of the fungal structures in the host appeared to be identical to that of the wild type. GFP accumulation in inoculated leaves of N. benthamiana was quantified in leaf extracts using a fluorescence microplate reader, and the quantity of fluorescence was strongly correlated with the growth of the fungus as measured by the amount of fungal actin gene expression using Northern blot hybridizations. These results demonstrated that assaying green fluorescence levels from a GFP-transformed fungus is an accurate, fast and easy means of quantifying fungal growth inside host plant cells.     

MAPK signaling regulates nitric oxide and NADPH oxidase-dependent oxidative bursts in Nicotiana benthamiana

Nitric oxide (NO) and reactive oxygen species (ROS) act as signals in innate immunity in plants. The radical burst is induced by INF1 elicitin, produced by the oomycete pathogen Phytophthora infestans. NO ASSOCIATED1 (NOA1) and NADPH oxidase participate in the radical burst. Here, we show that mitogen-activated protein kinase (MAPK) cascades MEK2-SIPK/NTF4 and MEK1-NTF6 participate in the regulation of the radical burst. NO generation was induced by conditional activation of SIPK/NTF4, but not by NTF6, in Nicotiana benthamiana leaves. INF1- and SIPK/NTF4-mediated NO bursts were compromised by the knockdown of NOA1. However, ROS generation was induced by either SIPK/NTF4 or NTF6. INF1- and MAPK-mediated ROS generation was eliminated by silencing Respiratory Burst Oxidase Homolog B (RBOHB), an inducible form of the NADPH oxidase. INF1-induced expression of RBOHB was compromised in SIPK/NTF4/NTF6-silenced leaves. These results indicated that INF1 regulates NOA1-mediated NO and RBOHB-dependent ROS generation through MAPK cascades. NOA1 silencing induced high susceptibility to Colletotrichum orbiculare but not to P. infestans; conversely, RBOHB silencing decreased resistance to P. infestans but not to C. orbiculare. These results indicate that the effects of the radical burst on the defense response appear to be diverse in plant-pathogen interactions.     

Possible involvement of maize Rop1 in the defence responses of plants to viral infection

The expression of host genes can be altered during the process of viral infection. To investigate the viral infection-induced up-regulated gene expression changes of maize at different time intervals post-inoculation with Sugarcane mosaic virus (SCMV), a suppression subtractive hybridization cDNA library was constructed. A total of 454 cDNA clones were identified to be viral infection-induced up-regulated genes. The influence of Rop1 on the infection of maize by SCMV was investigated. The results showed that transient silencing of the ZmRop1 gene through virus-induced gene silencing enhanced the accumulation and systemic infection of SCMV and another potyvirus (Pennisetum mosaic virus) in maize plants, whereas transient over-expression of ZmRop1 in maize protoplasts reduced SCMV accumulation. Furthermore, it was demonstrated that the heterologous expression of ZmRop1 impaired Potato virus X infection in Nicotiana benthamiana plants. These data suggest that ZmRop1 may play a role in plant defence responses to viral infection.     

The role of ethylene during the infection of Nicotiana tabacum by Colletotrichum destructivum

Two periods of increased ethylene production were observed after inoculation of Nicotiana tabacum by Colletotrichum destructivum. This pathogen exhibits an intracellular hemibiotrophic infection process, with a biotrophic phase followed by a necrotrophic phase. Ethylene production first increased during the biotrophic phase with a peak at 24 h before the necrotrophic phase. A second increase in ethylene occurred late in the necrotrophic phase when the lesions were expanding. Two different 1-aminocyclopropane-1-carboxylic acid synthase genes showed increased expression after the first ethylene peak with a maximum at 24 h before the second ethylene increase. Expression of an 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) gene increased during the first ethylene peak and then declined at the beginning of the second ethylene increase. A second ACO gene showed relatively little change in expression during infection with slightly higher expression at 24 h before the second ethylene increase, and a third ACO gene showed a progressive decline in expression with a major decrease occurring before the second ethylene increase. Inoculation of ethylene-insensitive tobacco with C. destructivum revealed that it was more susceptible than the wild type. The changes in ethylene production and associated gene expression as well as the increased disease susceptibility of ethylene-insensitive tobacco indicate that ethylene plays a role in this interaction, perhaps as a signalling molecule to trigger defense mechanisms.     

假马鞭曲叶病毒和中国胜红蓟黄脉病毒C4蛋白是RNA沉默的抑制子

为了研究中国胜红蓟黄脉病毒(Ageratum yellow vein Chin virus,AYVCNV)和假马鞭曲叶病毒(Stachytarpheta leafcurl virus,StaLCV)C4蛋白的功能,利用烟草脆裂病毒(Tobacco rattle virus,TRV)载体在本氏烟(Nicotianabenthamiana)中分别表达了这两种病毒的C4蛋白,结果发现它们均能在本氏烟中引起类似于病毒侵染的症状,推测AYVCNV和StaLCV的C4蛋白是病毒的致病因子;在RNA沉默的抑制试验中,AYVCNV和StaLCV的C4蛋白均能够在表达gfp基因的转基因本氏烟(16c)上抑制由gfp基因正义链引起的基因沉默的建立,证明它们都是RNA沉默的抑制子。     

Long-distance movement,virulence, and RNA silencing suppression controlled by a single protein in hordei- and potyviruses: complementary functions between virus families

RNA silencing is a natural defense mechanism against genetic stress factors, including viruses. A mutant hordeivirus (Barley stripe mosaic virus [BSMV]) lacking the gammab gene was confined to inoculated leaves in Nicotiana benthamiana, but systemic infection was observed in transgenic N. benthamiana expressing the potyviral silencing suppressor protein HCpro, suggesting that the gammab protein may be a long-distance movement factor and have antisilencing activity. This was shown for gammab proteins of both BSMV and Poa semilatent virus (PSLV), a related hordeivirus. Besides the functions in RNA silencing suppression, gammab and HCpro had analogous effects on symptoms induced by the hordeiviruses. Severe BSMV-induced symptoms were correlated with high HCpro concentrations in the HCpro-transgenic plants, and substitution of the gammab cistron of BSMV with that of PSLV led to greatly increased symptom severity and an altered pattern of viral gene expression. The efficient systemic infection with the chimera was followed by the development of dark green islands (localized recovery from infection) in leaves and exemption of new developing leaves from infection. Recovery and the accumulation of short RNAs diagnostic of RNA silencing in the recovered tissues in wild-type N. benthamiana were suppressed in HCpro-transgenic plants. These results provide evidence that potyviral HCpro and hordeivirus gammab proteins contribute to systemic viral infection, symptom severity, and RNA silencing suppression. HCpro's ability to suppress the recovery of plants from viral infection emphasizes recovery as a manifestation of RNA silencing.     

Salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by Agrobacterium tumefaciens

We investigated the effects of salicylic acid (SA) and systemic acquired resistance (SAR) on crown gall disease caused by Agrobacterium tumefaciens. Nicotiana benthamiana plants treated with SA showed decreased susceptibility to Agrobacterium infection. Exogenous application of SA to Agrobacterium cultures decreased its growth, virulence, and attachment to plant cells. Using Agrobacterium whole-genome microarrays, we characterized the direct effects of SA on bacterial gene expression and showed that SA inhibits induction of virulence (vir) genes and the repABC operon, and differentially regulates the expression of many other sets of genes. Using virus-induced gene silencing, we further demonstrate that plant genes involved in SA biosynthesis and signaling are important determinants for Agrobacterium infectivity on plants. Silencing of ICS (isochorismate synthase), NPR1 (nonexpresser of pathogenesis-related gene 1), and SABP2 (SA-binding protein 2) in N. benthamiana enhanced Agrobacterium infection. Moreover, plants treated with benzo-(1,2,3)-thiadiazole-7-carbothioic acid, a potent inducer of SAR, showed reduced disease symptoms. Our data suggest that SA and SAR both play a major role in retarding Agrobacterium infectivity.     

Virus-induced gene silencing of argonaute genes in Nicotiana benthamiana demonstrates that extensive systemic silencing requires Argonaute1-like and Argonaute4-like genes

Several distinct pathways of RNA silencing operate in plants with roles including the suppression of virus accumulation, control of endogenous gene expression, and direction of DNA and chromatin modifications. Proteins of the Dicer-Like and Argonaute (AGO) families have key roles within these silencing pathways and have distinct biochemical properties. We are interested in the relationships between different silencing pathways and have used Nicotiana benthamiana as a model system. While not being an amenable plant for traditional genetics, N. benthamiana is extensively used for RNA-silencing studies. Using virus-induced gene silencing technology we demonstrate that both NbAGO1- and NbAGO4-like genes are required for full systemic silencing but not for silencing directed by an inverted repeat transgene. Phenotypic differences between the virus-induced gene silencing plants indicate that NbAGO1 and NbAGO4 like act at different stages of the silencing pathways. Suppression of NbAGO1 expression recapitulated the hypomorphic mutant phenotype of certain Arabidopsis (Arabidopsis thaliana) ago1 alleles, however, suppression of NbAgo4 like resulted in phenotypes differing in some respects from those reported for Arabidopsis ago4. We suggest that the small interfering RNA amplification step required for full systemic silencing is dependent upon a nuclear event requiring the activity of NbAGO4 like.     

A novel Arabidopsis-Colletotrichum pathosystem for the molecular dissection of plant-fungal interactions

The ability of a Colletotrichum sp., originally isolated from Brassica campestris, to infect Arabidopsis thaliana was examined. Sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal (r)DNA showed the pathogen to be Colletotrichum destructivum. The host range was broad, including many cruciferous plants and some legumes. At 25 degrees C, all A. thaliana accessions tested were susceptible to the Brassica isolates of C. destructivum; however, at 15 degrees C, the accession Ws-2 showed a temperature-dependant resistance, in which single epidermal cells underwent a rapid hypersensitive response. Legume isolates of C. destructivum were unable to infect A. thaliana and induced deposition of callose papillae at sites of attempted penetration. In compatible interactions, C. destructivum showed a two-stage, hemibiotrophic infection process. The initial biotrophic phase was associated with large, intracellular primary hyphae and was confined to one epidermal cell; whereas, in the subsequent necrotrophic phase, narrow secondary hyphae extensively colonized the tissue and conidia were produced in acervuli. An efficient transformation system was established for C. destructivum, using Agrobacterium-mediated transfer of DNA. The ability to genetically manipulate both partners in the interaction is an important advantage, and the Arabidopsis-Colletotrichum pathosystem should provide a valuable new model for dissecting plant-fungal interactions.     

Optimization of transient Agrobacterium-mediated gene expression system in leaves of Nicotiana benthamiana

Here we report on a simple and reproducible system of Agrobacterium-mediated transient gene expression assay that utilizes infiltration of young Nicotiana benthamiana leaves. Although some of the phenomena described in this paper have been already reported by other researchers, here we have further developed them. The highest level of transient gfp gene expression was detected in the youngest leaves of N. benthamiana infiltrated with A. tumefaciens strains AGL0 and EHA105 precultured in the presence of 450-600 microM acetosyringone. Although the maximum level of transient gfp gene expression was restricted presumably by RNA silencing, it was completely suppressed in the presence of the viral protein HC-Pro. The transient expression system described here can be used to identify new viral suppressors of RNA silencing, for detailed analysis of unidentified genes and for industrial production of proteins in plants as well.