Yule Liu

Autophagy Regulates Programmed Cell Death during the Plant Innate Immune Response

The plant innate immune response includes the hypersensitive response (HR), a form of programmed cell death (PCD). PCD must be restricted to infection sites to prevent the HR from playing a pathologic rather than protective role. Here we show that plant BECLIN 1, an ortholog of the yeast and mammalian autophagy gene ATG6/VPS30/beclin 1, functions to restrict HR PCD to infection sites. Initiation of HR PCD is normal in BECLIN 1-deficient plants, but remarkably, healthy uninfected tissue adjacent to HR lesions and leaves distal to the inoculated leaf undergo unrestricted PCD. In the HR PCD response, autophagy is induced in both pathogen-infected cells and distal uninfected cells; this is reduced in BECLIN 1-deficient plants. The restriction of HR PCD also requires orthologs of other autophagy-related genes including PI3K/VPS34, ATG3, and ATG7. Thus, the evolutionarily conserved autophagy pathway plays an essential role in plant innate immunity and negatively regulates PCD.

Role of SCF Ubiquitin-Ligase and the COP9 Signalosome in the N Gene–Mediated Resistance Response to Tobacco mosaic virus

The tobacco N gene confers resistance to Tobacco mosaic virus (TMV) and encodes a toll–interleukin-1 receptor/nucleotide binding/Leu-rich repeat class protein. Recent evidence indicates that the Nicotiana benthamiana Rar1 gene (NbRar1), which encodes a protein with a zinc finger motif called CHORD (Cys- and His-rich domain), is required for the function of N. To investigate the role of NbRar1 in plant defense, we identified its interaction partners. We show that the NbRar1 protein interacts with NbSGT1, a highly conserved component of the SCF (Skp1/Cullin/F-box protein)-type E3 ubiquitin ligase complex involved in protein degradation. In addition, we show that NbSGT1 interacts with NbSKP1. Suppression of NbSGT1 and NbSKP1 shows that these genes play an important role in the N-mediated resistance response to TMV. Both NbRar1 and NbSGT1 associate with the COP9 signalosome, another multiprotein complex involved in protein degradation via the ubiquitin-proteasome pathway. Silencing of the NbCOP9 signalosome also compromises N-mediated resistance to TMV. Our results reveal new roles for SCF and the COP9 signalosome in plant defense signaling.

Molecular chaperone Hsp90 associates with resistance protein N and its signaling proteins SGT1 and Rar1 to modulate an innate immune response in plants.

SGT1 and Rar1 are important signaling components of resistance (R) gene-mediated plant innate immune responses. Here we report that SGT1 and Rar1 associate with the molecular chaperone Hsp90. In addition, we show that Hsp90 associates with the resistance protein N that confers resistance to tobacco mosaic virus. This suggests that Hsp90-SGT1-Rar1 and R proteins might exist in one complex. Suppression of Hsp90 in Nicotiana benthamiana plants shows that it plays an important role in plant growth and development. In addition, Hsp90 suppression in NN plants compromises N-mediated resistance to tobacco mosaic virus. Our results reveal a new role for SGT1- and Rar1-associated chaperone machinery in R gene-mediated defense signaling.

The Jasmonate-ZIM Domain Proteins Interact with the R2R3-MYB Transcription Factors MYB21 and MYB24 to Affect Jasmonate-Regulated Stamen Development in Arabidopsis

Jasmonate is essential for diverse biological processes, including male fertility and plant defense in Arabidopsis. This work shows that the R2R3-MYB transcription factors MYB21 and MYB24 function as direct targets of JAZ proteins to mediate jasmonate-regulated stamen development. The Arabidopsis thaliana F-box protein CORONATINE INSENSITIVE1 (COI1) perceives jasmonate (JA) signals and subsequently targets the Jasmonate-ZIM domain proteins (JAZs) for degradation by the SCFCOI1-26S proteasome pathway to mediate various jasmonate-regulated processes, including fertility, root growth, anthocyanin accumulation, senescence, and defense. In this study, we screened JAZ-interacting proteins from an Arabidopsis cDNA library in the yeast two-hybrid system. MYB21 and MYB24, two R2R3-MYB transcription factors, were found to interact with JAZ1, JAZ8, and JAZ11 in yeast and in planta. Genetic and physiological experiments showed that the myb21 myb24 double mutant exhibited defects specifically in pollen maturation, anther dehiscence, and filament elongation leading to male sterility. Transgenic expression of MYB21 in the coi1-1 mutant was able to rescue male fertility partially but unable to recover JA-regulated root growth inhibition, anthocyanin accumulation, and plant defense. These results demonstrate that the R2R3-MYB transcription factors MYB21 and MYB24 function as direct targets of JAZs to regulate male fertility specifically. We speculate that JAZs interact with MYB21 and MYB24 to attenuate their transcriptional function; upon perception of JA signal, COI1 recruits JAZs to the SCFCOI1 complex for ubiquitination and degradation through the 26S proteasome; MYB21 and MYB24 are then released to activate expression of various genes essential for JA-regulated anther development and filament elongation.

Virus-Induced Gene Silencing

In the postgenomic era, large-scale functional genomic approaches are necessary for converting sequence information into functional information. A para-genetic approach, called virus-induced gene silencing (VIGS), offers a rapid means of gaining insight into gene function in plants. VIGS system could be used to suppress endogenous gene expression by infecting plants with a recombinant virus vector (VIGS vector) carrying host-derived sequence. Here, we describe the use of tobacco rattle virus (TRV)-based VIGS technique to study gene function in Nicotiana benthamiana and tomato.

Efficient Virus-Induced Gene Silencing in Arabidopsis

Virus-induced gene silencing (VIGS) is a plant RNA-silencing technique that uses viral vectors carrying a fragment of a gene of interest to generate double-stranded RNA, which initiates the silencing of the target gene. Several viral vectors have been developed for VIGS and they have been successfully used in reverse genetics studies of a variety of processes occurring in plants. This approach has not been widely adopted for the model dicotyledonous species Arabidopsis (Arabidopsis thaliana), possibly because, until now, there has been no easy protocol for effective VIGS in this species. Here, we show that a widely used tobacco rattle virus-based VIGS vector can be used for silencing genes in Arabidopsis ecotype Columbia-0. The protocol involves agroinfiltration of VIGS vectors carrying fragments of genes of interest into seedlings at the two- to three-leaf stage and requires minimal modification of existing protocols for VIGS with tobacco rattle virus vectors in other species like Nicotiana benthamiana and tomato (Lycopersicon esculentum). The method described here gives efficient silencing in Arabidopsis ecotype Columbia-0. We show that VIGS can be used to silence genes involved in general metabolism and defense and it is also effective at knocking down expression of highly expressed transgenes. A marker system to monitor the progress and efficiency of VIGS is also described.

Genome-Wide ORFeome Cloning and Analysis of Arabidopsis Transcription Factor Genes

Here, we report our effort in generating an ORFeome collection for the Arabidopsis transcription factor (TF) genes. In total, ORFeome clones representing 1,282 Arabidopsis TF genes have been obtained in the Gateway high throughput cloning pENTR vector, including 411 genes whose annotation lack cDNA support. All the ORFeome inserts have also been mobilized into a yeast expression destination vector, with an estimated 85% rate of expressing the respective proteins. Sequence analysis of these clones revealed that 34 of them did not match with either the reported cDNAs or current predicted open-reading-frame sequences. Among those, novel alternative splicing of TF gene transcripts is responsible for the observed differences in at least five genes. However, those alternative splicing events do not appear to be differentially regulated among distinct Arabidopsis tissues examined. Lastly, expression of those TF genes in 17 distinct Arabidopsis organ types and the cultured cells was profiled using a 70-mer oligo microarray.

The bHLH Transcription Factor MYC3 Interacts with the Jasmonate ZIM-Domain Proteins to Mediate Jasmonate Response in Arabidopsis

The Arabidopsis Jasmonate ZIM-domain proteins (JAZs) act as substrates of SCF(COI1) complex to repress their downstream targets, which are essential for JA-regulated plant development and defense. The bHLH transcription factor MYC2 was found to interact with JAZs and mediate JA responses including JA-inhibitory root growth. Here, we identified another bHLH transcription factor MYC3 which directly interacted with JAZs by virtue of its N-terminal region to regulate JA responses. The transgenic plants with overexpression of MYC3 exhibited hypersensitivity in JA-inhibitory root elongation and seedling development. The JAZ-interacting pattern and the JA-induced expression pattern of MYC3 were distinguishable from those of MYC2. We speculate that MYC3 and MYC2 may have redundant but also distinguishable functions in regulation of JA responses.

The Mi-1-Mediated Pest Resistance Requires Hsp90 and Sgt1

The tomato (Solanum lycopersicum) Mi-1 gene encodes a protein with putative coiled-coil nucleotide-binding site and leucine-rich repeat motifs. Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum euphorbiae), and sweet potato whitefly (Bemisia tabaci). To identify genes required in the Mi-1-mediated resistance to nematodes and aphids, we used tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to repress candidate genes and assay for nematode and aphid resistance. We targeted Sgt1 (suppressor of G-two allele of Skp1), Rar1 (required for Mla12 resistance), and Hsp90 (heat shock protein 90), which are known to participate early in resistance gene signaling pathways. Two Arabidopsis (Arabidopsis thaliana) Sgt1 genes exist and one has been implicated in disease resistance. Thus far the sequence of only one Sgt1 ortholog is known in tomato. To design gene-specific VIGS constructs, we cloned a second tomato Sgt1 gene, Sgt1-2. The gene-specific VIGS construct TRV-SlSgt1-1 resulted in lethality, while silencing Sgt1-2 using TRV-SlSgt1-2 did not result in lethal phenotype. Aphid and root-knot nematode assays of Sgt1-2-silenced plants indicated no role for Sgt1-2 in Mi-1-mediated resistance. A Nicotiana benthamiana Sgt1 VIGS construct silencing both Sgt1-1 and Sgt1-2 yielded live plants and identified a role for Sgt1 in Mi-1-mediated aphid resistance. Silencing of Rar1 did not affect Mi-1-mediated nematode and aphid resistance and demonstrated that Rar1 is not required for Mi-1 resistance. Silencing Hsp90-1 resulted in attenuation of Mi-1-mediated aphid and nematode resistance and indicated a role for Hsp90-1. The requirement for Sgt1 and Hsp90-1 in Mi-1-mediated resistance provides further evidence for common components in early resistance gene defense signaling against diverse pathogens and pests.

Virus induced gene silencing of a DEFICIENS ortholog in Nicotiana benthamiana.

Traditionally, developmental studies in plant biology have suffered from the lack of a convenient means to study gene function in non-model plant species. Here we show that virus-induced gene silencing (VIGS) is an effective new tool to study the function of orthologs of floral homeotic genes such as DEFICIENS(DEF) in non-model systems. We used a tobacco rattle virus (TRV)-based VIGS approach to study the function of the Nicotiana benthamiana DEFortholog (NbDEF). Silencing of NbDEFin N. benthamianausing TRV-VIGS was similar to that of Antirrhinum defand Arabidopsis ap3mutants and caused transformation of petals into sepals and stamens into carpels. Molecular analysis of the NbDEF-silenced plants revealed a dramatic reduction of the levels of NbDEFmRNA and protein in flowers. NbDEFsilencing was specific and has no effect on the mRNA levels of NbTM6, the closest paralog of NbDEF. A dramatic reduction of the levels of N. benthamiana GLOBOSA(NbGLO) mRNA and protein was also observed in flowers of NbDEF-silenced plants, suggesting that cross-regulation of this GLO-like gene by NbDEF. Taken together, our results suggest that NbDEF is a functional homolog of Antirrhinum DEF. Our results are significant in that they show that TRV efficiently induces gene silencing in young and differentiating flowers and that VIGS is a promising new tool for analyses of developmental gene function in non-model organisms.