BACKGROUND
unlike animals, plants are sessile and must overcome or endure variable, sometimes severe environmental conditions.
for example, plants have evolved two main defense mechanisms, pathogen-associated molecular pattern -triggered immunity  and effecter-triggered immunity , to combat pathogen infections  <cit> .
eti is a much stronger reaction than pti and is often accompanied by a localized hypersensitive response , a type of programmed cell death , to capture the pathogen at the site of infection.
autophagy is an evolutionarily conserved, eukaryotic process by which organelles and cytosolic macromolecules are consumed in lysosomes  for nutrient recycling  <cit> .
recent evidence from autophagy-defective arabidopsis plants suggests an important role for autophagy in regulating plant immune responses  <cit> .
this role, however, remains unclear since both positive and negative effects of autophagy on plant immunity have been described in different arabidopsis-pathogen systems as well as in the same arabidopsis-pathogen system  <cit> .
likewise, both pro-survival and pro-death roles of autophagy in the regulation of hr-pcd or pathogen-induced cell death have been reported  <cit> .
therefore, the mechanisms behind the relationship between autophagy and plant immunity need to be elucidated in detail when considering plant species other than arabidopsis and more plant-pathogen systems.
during autophagy, double-membrane structures called autophagosomes facilitate the transport of cellular cargos into lysosomes for degradation  <cit> .
to date, more than  <dig> autophagy-related proteins  functioning in autophagy induction and regulation, the initiation and biogenesis of autophagosomes, and the fusion of autophagosomes with lysosomes have been identified in yeast and some orthologs identified in animals and plants  <cit> .
in yeast, a class iii phosphatidylinositol 3-kinase  complex resides in the pre-autophagosomal structure/phagophore assembly site  where it catalyzes phosphatidylinositol 3-phosphate  synthesis and recruits pi3p-binding proteins, especially the atg18-atg <dig> complex, for the initiation of autophagic membranes  <cit> .
yeast atg6/vacuolar protein sorting  <dig> , the ortholog of mammalian beclin  <dig>  is the core component of the pi3k complex along with vps <dig>  vps <dig> and atg <dig>  <cit>  and is essential for auophagy.
plant homologs of yeast atg <dig> play similar essential roles in autophagy, for atg6-deficient plants produce a reduced number of autolysosomes under autophagy-inducing conditions  <cit> , and the expression of plant atg <dig> restores autophagy in atg6-mutant yeast  <cit> .
atg6-deficient plants, like mutants of other atg genes, show an enhanced sensitivity to nutrient starvation and other stress factors  <cit> .
this enhanced sensitivity may result from impaired autophagy because autophagy is responsible for nutrient recycling and distribution under severe environmental conditions.
however, the atg6-associated pi3k complex is not autophagy-specific.
its phospholipid product, pi3p, is a well-known second messenger involved in receptor signaling and protein sorting-related vesicle trafficking events  <cit> .
homozygous plant mutants of atg <dig>  vps <dig> or vps <dig> cannot be obtained because of the requirement of pi3k in pollen development, germination and pollen tube growth  <cit> .
such effects may be attributed to the pi3p-, or its derivative pip2-, signaling pathways rather than to autophagy because homozygous mutants of the autophagy-specific atg genes can be routinely obtained by selfing heterozygous individuals.
atg6-deficient plants also show more severe growth abnormalities under normal conditions than mutants of the autophagy-specific atg genes, although they have similarly reduced autophagy levels  <cit> .
atg <dig>  like autophagy-specific factors, is implicated in plant immune responses  <cit> .
yet the pleiotropy of atg <dig> makes it difficult to interpret whether autophagy, the pi3p-signaling pathways, or their interplay underlines the roles of atg <dig> in plant immunity.
well-established roles of atg <dig>  other than autophagy, also include its involvement in the vacuolar protein sorting pathway of yeast and in tumor suppression through interactions with the anti-apoptotic bcl-2-like proteins in mammals  <cit> .
so far, few studies on the mechanisms and physiological functions of autophagy and other atg6-associated pathways have been reported in crop plants, and fewer still in common wheat , which is an important food crop worldwide.
here, we report the identification of three members of the wheat atg <dig> family.
the conserved function of wheat atg6s in autophagy was shown using yeast functional complementation, subcellular localization of green fluorescent protein  fusions and the determination of autophagy levels in atg <dig> knockdown plants.
a role for atg <dig> in the wheat immune response to the causal fungus, blumeria graminis f. sp.
tritici , of powdery mildew was established by bgt-induced atg <dig> expression profiles and by altered powdery mildew symptoms on atg <dig> knockdown plants.
methods
plant materials and fungal strain
wheat used in this study included isogenic lines 92r137/yangmai  <dig> and yangmai  <dig>  and isogenic lines michigan amber/chancellor <dig> and chancellor.
92r137/yangmai  <dig> carries the broad-spectrum powdery mildew resistance gene pm <dig>  michigan amber/chancellor <dig> carries the isolate-specific powdery mildew resistance gene pm3f.
yangmai  <dig> and chancellor are susceptible to powdery mildew.
the prevalent chinese bgt isolate e <dig>  which is avirulent to pm <dig> and pm3f, was maintained on seedlings of the susceptible wheat cultivar sumai  <dig> under spore-proof conditions.
plant growth conditions and treatments
all wheat plants were cultured hydroponically in one-fifth strength hoagland’s solution under normal conditions of 22°c/18°c  and a photoperiod of 16 h light/8 h dark in controlled climate chambers.
two-leaf stage seedlings were subjected to different treatments.
pathogen inoculation was conducted by vigorously shaking off fresh bgt conidia from diseased sumai  <dig> plants onto experimental seedlings.
for abiotic stress treatments, seedlings were transferred to the same one-fifth strength hoagland’s solution containing 200 mm nacl  or 16% peg <dig> .
additional abiotic stress treatments were nitrogen deprivation, and low temperature combined with darkness, in which seedlings were transferred to a 4°c refrigerator and kept in the dark.
leaves harvested at defined time points were frozen with liquid nitrogen and then stored at −80°c.
these leaf samples were used for gene isolation and expression analyses.
isolation of wheat atg <dig> genes and expression analyses
total rna was prepared from each harvested leaf sample using trizol  and subjected to rnase-free dnase i digestion and purification.
first-strand cdna was synthesized using oligo primers and the quantscript rt kit .
the protein sequence of arabidopsis atg <dig>  was used as the input to search for wheat expressed sequence tags  in the dbest division of genbank by tblastn.
both 5ʹ- and 3ʹ-terminal ests were iteratively used as queries to search for new terminal ests to cover full-length open reading frames .
est contigs were assembled in vector nti advance  <dig> , and their sequences were used to design primers  flanking full-length orfs.
atg <dig> genes were amplified from first-strand cdna using the high-fidelity pfu dna polymerase.
amplified products were a-tailed and then cloned into the vector pgem®-t easy  for sequencing.
sequence data were deposited in the genbank database.
the expression patterns of wheat atg6s responding to biotic and abiotic treatments were quantified by quantitative real-time pcr  with gene-specific primers .
qrt-pcr was conducted in a 20 μl realmastermix  mixture  containing 1 μl first-strand cdna as the template, and the reaction was performed on an iq <dig> .
each cdna sample was analyzed in triplicate.
relative expression levels were calculated using the 2-ΔΔct method  <cit>  with the amplicon of the wheat β-tubulin gene as an internal control.
chromosomal localization of wheat atg6s
wheat nullisomic-tetrasomic lines  were used to map atg <dig> genes on specific chromosomes.
dna of each line was isolated and used as the template for pcr amplification with gene-specific primers .
amplified products were analyzed by 12% polyacrylamide gel electrophoresis.
chromosome localization was inferred based on the absence of amplified fragments from lines lacking specific chromosomes.
complementation test of yeast atg <dig> mutant
the orf of each wheat atg <dig> cdna was amplified as a noti- noti fragment using the primers listed in additional file 2: table s <dig>  amplified products were digested and cloned into the yeast expression vector pfl <dig>  in which the constitutive expression of the inserted gene was driven by the phosphoglycinecerate kinase  gene promoter.
constructed vectors were sequenced to confirm the correct cdna insertion direction.
wild-type yeast strain by <dig> and a mutant strain, atg <dig> , were purchased from open biosystems .
each constructed vector was introduced into yeast atg <dig> cells according to the liac/ss-dna/peg trafo protocol  <cit> .
positive transformants were screened on sc-u medium.
the yeast autophagy complementation test was performed as previously described  <cit> .
the accumulation of autophagic bodies in vacuoles was observed under a differential interference contrast microscope .
subcellular localization of wheat atg6s
the orf of each wheat atg <dig> cdna lacking a stop codon was amplified as an xhoi-spei fragment using primers listed in additional file 2: table s <dig>  amplified products were digested, and ligated upstream and in-frame with the orf of gfp in the vector pa7-gfp, in which the gfp fusion construct’s expression was driven by two tandem 35s promoters.
prepared plasmids were introduced into onion epidermal cells by particle bombardment with pds1000/he .
gfp fluorescence was visualized by epifluorescence microscopy  24 h after bombardment.
virus-induced gene silencing
a barley stripe mosaic virus -based vigs method  <cit>  was used to create gene knockdown plants.
to maximize knockdown efficiency, three bsmvγ vectors, each containing a 200–241 bp fragment from the upstream, middle, or downstream region of the taatg6a coding sequence, were constructed, and in vitro transcribed rnaγ molecules from these vectors were premixed for virus infection.
to circumvent the potential functional redundancy among paralogs, we intended to produce simultaneous knockdowns of wheat atg <dig> members by choosing fragments from taatg6a for the construction of bsmvγ vectors with  <dig> – <dig> % sequence identities to the corresponding fragments of taatg6b and 6c and multiple nucleotide stretches longer than 23 bp that are 100% identical among the three atg <dig> members.
the three fragments were prepared by pcr using the primers containing nhei sites listed in additional file 2: table s <dig>  each amplified fragment was digested by nhei and then inserted in the reverse orientation into the vector bsmvγ to produce the vectors bsmvγ:taatg6as- <dig>  bsmvγ:taatg6as- <dig> and bsmvγ:taatg6as- <dig>  plasmids of these three bsmvγ derivatives, as well as bsmvγ:gfp and bsmvα, were linearized by mlui, and bsmvβ was linearized by spei.
linearized vectors were in vitro transcribed to produce 5ʹ-capped infectious bsmv rna molecules using the ribomax large scale rna production-t <dig> kit  with the addition of a cap analog  in the transcription mixture.
rnaγ:taatg6as- <dig>  rnaγ:taatg6as- <dig>  and rnaγ:taatg6as- <dig> were premixed in a 1:1: <dig> ratio to produce rnaγ:taatg6as.
the second fully expanded leaves of wheat seedlings at the two-leaf stage were mechanically infected with a 1:1: <dig> mixture of rnaα, rnaβ and rnaγ:taatg6as  in 1× gkp buffer  <cit> .
seedlings infected with a 1:1: <dig> mixture of rnaα, rnaβ and rnaγ:gfp  in 1× gkp buffer were used as controls.
three replications of the vigs experiment were conducted on the two isogenic lines of 92r137/yangmai  <dig> and yangmai  <dig>  in each replication, a total of  <dig> seedlings per line were used for each of the bsmv:atg <dig> and bsmv:gfp treatments.
the bsmv-treated plants were maintained on one-fifth strength hoagland’s solution under normal conditions.
the success of the virus infection was judged by visual observations of mild chlorotic mosaic symptoms on the third and upper leaves of bsmv-treated plants.
to confirm the knockdown efficiency, eight plants with virus symptoms were randomly selected from each of the two bsmv treatments, and their transcript levels of taatg6s in the fourth leaves were determined using qrt-pcr as described above with a primer pair  designed to conserve regions in the three wheat atg6s.
determination of autophagy level by lysotracker red staining
bsmv-treated plants at the four-leaf stage were inoculated with bgt conidia as described above.
the fourth leaves were excised at  <dig> and 7 days after inoculation  with bgt, infiltrated with 100 μm e-64d  and then stained with 2 μm lysotracker red dnd- <dig>  according to the procedure described previously  <cit> .
fluorescence signals were observed under a confocal microscope  using a 543-nm excitation laser and  <dig> to 615-nm band pass.
chlorophyll content determination
the fifth leaves of bsmv-treated plants at the five-leaf stage were excised, weighed and ground in 1 ml of 80%  acetone.
the chlorophyll content was determined spectrophotometrically according to patel and dinesh-kumar  <cit> .
leaf senescence assay
green sections were excised from the fourth leaves of bsmv-treated plants at the four-leaf stage and floated on deionized water at room temperature in the dark.
leaf sections were photographed every day after dark incubation.
the experiment was repeated in triplicate using leaves of three or more plants in each experiment.
evaluation of powdery mildew resistance
to exclude the influence of early senescence/chlorosis on bgt infections, only those bsmv:atg6-treated plants without obvious symptoms of early senescence and chlorosis were selected for resistance evaluation.
bsmv-treated plants at the six-leaf stage were inoculated with bgt conidia as described above.
for bsmv-treated yangmai  <dig> plants, leaf discs with visible mildew colonies were gently harvested from different regions of the fourth leaves at  <dig> dai, immersed in  <dig> % glycerol and subjected to an ultrasonic treatment for 1 min to disperse the conidia.
the conidial production per mildew colony was counted by microscopic observation and expressed as the mean ± sd of at least  <dig> mildew colonies from five plants.
fungal structures were stained by trypan blue  <cit>  and visualized by light microscopy.
for bsmv-treated 92r137/yangmai  <dig>  plants, the number of successfully penetrating conidium with secondary hyphae on the fourth leaves was counted at  <dig> dai and expressed as a percentage of at least  <dig> interaction sites on individual plants.
the number of secondary hypha associated with each successfully penetrating conidium was also counted and expressed as the mean ± sd of the observed successfully penetrating conidia on all of the investigated plants.
for bsmv-treated yangmai  <dig> plants, the number of mycelium with mature chains of conidia on the fourth leaves was counted at  <dig> dai and expressed as a percentage of at least  <dig> mycelia on the leaves of five plants.
statistical analysis
all experiments were repeated three times with similar results.
quantitative data were statistically analyzed using student’s t test  using the ibm spss <dig>  software package.
RESULTS
isolation of atg <dig> homologs from wheat
using the sequence of arabidopsis atg <dig> as a query,  <dig> putative wheat atg6-encoding ests were mined from the dbest division of genbank and assembled into contigs.
primers covering the full-length orfs of these contigs were designed and used for gene isolation by pcr from a leaf cdna pool of the 92r137/yangmai  <dig>  plants induced by bgt for 48 h. three genes, which encoded a typical atg <dig> domain  , were identified as atg <dig> homologs in wheat and designated as taatg6a , 6b  and 6c .
the orf nucleotide sequences of taatg6a, 6b and 6c share identities of  <dig> – <dig> % and encode peptide sequences of  <dig>   <dig>  and  <dig> amino acids, respectively, with 98–99% similarity.
in the eukaryotic atg <dig> phylogenetic tree , plant atg6s are separately grouped into monocot and dicot clades.
taatg6a-6c showed 90–93% sequence similarities to osatg6a-6c from rice and 75% to atatg <dig> from arabidopsis.
the atg <dig> domain region is more conserved across all plant atg6s , whereas the n- and c-terminal flanking sequences show divergence between the monocot and dicot atg6s .
two methods, gene-specific pcr amplification of wheat ntl dnas and blast searches against the mapped wheat genomic sequences   <cit> , were performed to determine the chromosomal localizations of wheat atg <dig> members.
taatg6b and 6c were localized on chromosomes 3bl and 3al, respectively, by both methods .
taatg6a was localized on chromosome 3dl based only on the results of blast searches.figure  <dig>
chromosomal localization of wheat autophagy-related atg <dig> genes.
dna of each wheat nullisomic-tetrasomic line was used as a template for pcr amplification with gene-specific primers.
amplified products were analyzed on 12% polyacrylamide gels.
chromosome localization was inferred based on the absence of amplified fragments  in lines lacking the specific chromosomes.
we also obtained the respective genomic sequences of taatg6a, 6b and 6c by blast searches against wheat genomic sequences.
similar nine-intron structures of the taatg6a-6c orf sequences were revealed when comparing genomic sequences with the corresponding cdna sequences .
however, taatg6b has a much longer intron  <dig> than taatg6a and 6c.
we compared the three genomic sequences and found a 900-bp insertion only in the intron  <dig> of taatg6b, which is bordered by 19-bp direct short sequence repeats  .
as it encodes a truncated protein similar to rna-directed dna polymerases and polyproteins of mobile elements and because no long terminal repeats  were found around it, this inserted sequence most likely resulted from a transposition event of a non-ltr retrotransposon.
a cpg island of 999 bp, covering the start codon, was detected in the taatg6b genomic sequence, which has a  <dig> % g + c content and a ratio of  <dig>  of the observed versus expected presence of cpg dinucleotides .
this cpg island exists in other monocot atg6s as well, including osatg6b from rice, but not in dicot atg6s, such as atatg <dig> from arabidopsis .
within the cpg island, there is a short gc-rich stretch just downstream of the start codon, and its length varies among paralogs as well as among homologs .
only insertions or deletions that involve multiples of three bases are retained in the gc-rich stretch of monocot atg6s because they do not result in translational frame shifts.
amino acids encoded in the gc-rich stretch are predominantly glycines and alanines.figure  <dig>
characterization of wheat autophagy-related
atg6
sequences.
the genomic sequences of monocot atg6s contain a cpg island covering the start codon.
the cpg island was predicted using the web tool cpg island searcher  with a cpg island standard of longer than 500 bp in length, a minimum g + c content of 55% and a minimum  <dig>  ratio of the observed versus the expected presence of cpg dinucleotides.
gray, horizontal lines represent the 1600-bp start codon-covering genomic sequences of taatg6b from wheat, osatg6b  from rice and atatg <dig>  from arabidopsis.
short vertical red lines represent cpg dinucleotides.
blue horizontal thick lines represent the ranges of predicted cpg islands.
short gc-rich stretch just downstream of the start codon in monocot atg <dig> sequences.
sequences shown are the 5ʹ-terminal cdna sequences beginning with the start codons of monocot atg6s, including taatg6a, 6b and 6c from wheat, am <dig> from hordeum vulgare, nm_ <dig>  ct <dig> and nm_ <dig> from rice, xm_ <dig> from brachypodium distachyon, xm_ <dig> from oryza brachyantha, nm_ <dig> from zea mays, xm_ <dig> and am <dig> from sorghum bicolor, am <dig> from saccharum officinarum, and xm_ <dig> and xm_ <dig> from setaria italic.
the gc-rich stretches  are not properly aligned but the flanking sequences are aligned.
the reading frames from atg are indicated by the upper vertical lines.
wheat atg6s function in autophagy
human and arabidopsis atg <dig> can rescue the autophagy activity of atg6-mutant yeast  <cit> .
to determine if wheat atg6s are functional homologs of yeast atg <dig>  yeast expression vectors were constructed for each taatg <dig> and transformed into atg6-mutant yeast cells.
autophagic bodies in vacuoles were monitored as an indicator of autophagy activity.
after 5 h of nutrient starvation and treatment with the protease inhibitor phenylmethylsulfonyl fluoride, the wild-type yeast cells accumulated numerous autophagic bodies in vacuoles, which were rarely observed in the atg6-mutant yeast cells .
cells of atg6-mutant yeast transformed with taatg6a, 6b or 6c partially restored the accumulation of autophagic bodies .figure  <dig>
autophagy-related atg <dig> genes function in wheat autophagy.
functional complementation of atg6-mutant yeast cells by wheat atg6s.
the autophagic bodies accumulated within vacuoles are indicated by arrows.
scale bars represent 5 μm.
subcellular localization of gfp-taatg <dig> fusion structures in onion epidermal cells.
scale bars represent 50 μm.
localization of gfp-taatg6a in cytoplasmic and perinuclear membrane-like structures in onion epidermal cells.
florescence spots localized in cytoplasmic and perinuclear membrane-like structures are indicated by arrows.
scale bars represent 10 μm.
relative transcript levels of wheat atg6s in leaves of barley stripe mosaic virus -treated plants.
the fourth leaves were sampled from bsmv-treated 92r137/yangmai  <dig>  plants under normal conditions and used in the quantification of taatg <dig> transcripts by qrt-pcr with a primer pair designed based on identical regions of taatg6a, 6b, and 6c.
amplification of the wheat β-tubulin gene served as an internal control.
ck is from bsmv:gfp-treated control plants and arabic numbers indicate individual bsmv:atg6-treated plants.
constitutive and blumeria graminis f. sp.
tritici -induced autophagy are impaired in bsmv:atg6-treated 92r137/yangmai  <dig>  plants.
the fourth leaves were excised from bsmv-treated plants at  <dig> and 7 days after inoculation  with bgt conidia, infiltrated with e-64d and then stained with lysotracker red.
lysotracker red-stained punctate autolysosome-like structures are indicated by arrows.
the results were reproduced from three independent experiments using three or more plants in each experiment.
scale bars represent 5 μm.
punctate structures of atg6-gfp fluorescence, considered as pas for autophagosome initiation, have been observed in cells of yeast and arabidopsis  <cit> .
here, gfp fusion constructs of wheat atg6s were introduced into onion epidermal cells by particle bombardment.
punctate florescence signals were clearly observed in the cytoplasm of cells expressing taatg6a-gfp and seemed not to occur in the central vacuole .
fluorescent spots of taatg6a-gfp were sometimes clustered at cytoplasmic and perinuclear membrane-like structures resembling the trans-golgi network and endoplasmic reticulum .
localizations of atg6s to endosomes, the trans-golgi network, endoplasmic reticulum, mitochondria, cytoplasm, and nucleus have been reported in yeast and human  <cit> .
unexpectedly, taatg6b- and taatg6c-gfp florescence showed diffused distributions in the cytoplasm and nuclei, which were almost indistinguishable from those in cells expressing gfp alone .
therefore, additional signals may be required to trigger the translocation of taatg6b and 6c to pas for their role in autophagy initiation.
to determine if an atg <dig> deficiency can lead to autophagy impairment in wheat, bsmv:atg6-treated taatg <dig> knockdown plants were generated by the vigs method and their autophagy levels were compared with bsmv:gfp-treated control plants.
to obtain the maximum knockdown efficiency, bsmv:atg <dig> was designed to contain a mix of three bsmv rnaγ molecules, each containing an inserted antisense fragment corresponding to the upstream, middle, and downstream regions of the taatg6a coding sequence.
taking into consideration the potential functional redundancy among paralogs in polyploid wheat, bsmv:atg <dig> was also designed to knock down all three wheat atg <dig> members by inserting three fragments in the rnaγ molecules that are highly conserved  among the three genes.
as determined by qrt-pcr using a primer pair designed based on identical regions of the three taatg6s, the total constitutive transcript levels of taatg6a-6c were greatly reduced in the bsmv:atg6-treated plants compared with the transcript levels in bsmv:gfp-treated control plants .
this high knockdown efficiency was achieved in all eight bsmv:atg6-treated plants investigated, which confirmed the effectiveness of our vigs design.
we then inoculated bsmv-treated plants with bgt conidia and determined their constitutive autophagy activity at  <dig> dai and bgt-induced autophagy activity at  <dig> dai using the lysotracker red staining method.
lysotracker red is a dye that stains acidic organelles, such as lysosomes and endosomes, as well as autophagosomes/autolysosomes .
punctate structures labeled by this dye are generally considered indicative of plant autophagy activity  <cit> .
before lysotracker red staining, leaf samples were pre-infiltrated with the cysteine protease inhibitor e-64d to enhance the accumulation of autophagic bodies inside autolysosomes  <cit> .
for the bsmv:gfp-treated control plants of the resistant line 92r137/yangmai  <dig> , low levels of a lysotracker red-stained punctate pattern were observed at  <dig> dai, and enhanced levels were observed at  <dig> dai in bgt-challenged leaf epidermal cells .
these bgt-enhanced levels implied that the taatg6s-regulating autophagy process was induced and implicated in the pm21-triggered immune response to powdery mildew.
however, because of the knocking down of taatg6s, the lysotracker red-stained punctate structures were not visible in leaf samples of bsmv:atg6-treated plants regardless of bgt challenge .
this result of impaired autophagy in taatg <dig> knockdown plants, together with the complementation of yeast autophagy-defective cells with each taatg <dig> and the observation of pas localization of taatg6a-gfp, demonstrates the essential role of taatg6s in wheat autophagy.
wheat atg <dig> knockdown plants exhibited growth abnormalities
bsmv:atg6-treated plants, when compared with control plants, showed varying degrees of growth defects, including stunted growth and chlorosis under normal conditions .
some were thin and weak, and a higher percentage of bsmv:atg6-treated plants shifted into early senescence and accelerated death before the five-leaf stage than of the controls .
to further monitor senescence, green leaf sections from plants at the same developmental stage were excised and kept floating on water in the dark to induce senescence.
leaf sections from bsmv:atg6-treated plants began senescing within  <dig> d post-detachment, as indicated by yellowing .
this was significantly earlier than leaf sections from control plants, which began senescing by  <dig> d post-detachment.
thus, taatg6s are essential for normal plant growth and to prevent premature senescence in wheat.figure  <dig>
wheat autophagy-related
atg6
knockdown plants display growth abnormalities.
all barley stripe mosaic virus -treated plants were cultured hydroponically in one-fifth strength hoagland’s solution and kept under normal conditions of 22°c/18°c  and a photoperiod of 16 h light/8 h dark in controlled climate chambers.
the growth morphology of bsmv-treated yangmai  <dig> plants.
photographs were taken at the three-leaf stage.
the second leaves, which had been mechanically infected with bsmv, were generally withered by this stage and thus were excised before photographing.
leaf length of bsmv-treated yangmai  <dig> plants.
the lengths of the third leaves were measured at the three-leaf stage.
values represent the mean ± sd  of data measured from  <dig> individual plants.
asterisks indicate a significant difference  between the bsmv:atg6-treated plants and bsmv:gfp-treated control plants.
chlorophyll content of bsmv-treated yangmai  <dig> plants.
the chlorophyll content of the fifth leaves was determined at the five-leaf stage.
values represent the mean ± sd of data determined from five individual plants.
asterisks indicate a significant difference  between the bsmv:atg6-treated plants and bsmv:gfp-treated control plants.
percentage of bsmv-treated yangmai  <dig> plants displaying accelerated death before the five-leaf stage.
values represent the mean percentage ± sem  of at least  <dig> plants.
detached leaves from bsmv:atg6-treated plants display accelerated dark-induced senescence.
green sections of the fourth leaves were excised from bsmv-treated yangmai  <dig> plants at the four-leaf stage and were floated on deionized water at room temperature in the dark.
leaf sections were photographed at the indicated days post-detachment .
the results are reproduced from three independent experiments using the leaves of three or more plants in each experiment.
expression of wheat atg6s in response to abiotic stress factors and fungal infection
yangmai  <dig> plants were studied using qrt-pcr to determine the effects of abiotic stress factors on the expression of wheat atg6s.
the expression levels of taatg6b and 6c were upregulated by high salinity, drought, low temperature/darkness and nitrogen deprivation .
the expression of taatg6a was also upregulated by high salinity, drought and nitrogen deprivation, but downregulated by low temperature/darkness .
among the four investigated abiotic stress factors, wheat atg6s were most sensitive to the two osmotic stress factors, high salinity and drought, with up to 63-fold  and 230-fold  increases in the accumulation of transcripts.
these upregulated expression modes indicate that taatg6s are closely related to the wheat’s responses to abiotic stress factors.figure  <dig>
expression patterns of wheat autophagy-related atg <dig> genes.
two-leaf stage seedlings were subjected to different treatments.
samples were collected at the indicated time points after the initiation of treatment.
total rna was extracted and qrt-pcr was performed with gene-specific primers.
the relative expression levels were normalized by the wheat β-tubulin gene and relative to the control value measured at 0 h. data represent the mean of three independent experiments ± sd.
expression of wheat atg6s in response to abiotic stress factors.
yangmai  <dig> seedlings were transferred to the one-fifth strength hoagland’s solution containing 200 mm nacl  or 16% peg <dig> .
additional seedling treatments were nitrogen deprivation and exposure to 4°c in the dark .
expression of wheat atg6s in response to blumeria graminis f. sp.
tritici  infection.
pathogen inoculations were conducted by vigorously shaking fresh bgt conidia from diseased plants onto seedlings of the isogenic lines 92r137/yangmai  <dig>  and yangmai  <dig> , and the isogenic lines michigan amber/chancellor <dig>  and chancellor .
two powdery mildew resistant lines, 92r137/yangmai  <dig>  and michigan amber/chancellor <dig> , and their respective isogenic susceptible lines were included in the expression analysis, using qrt-pcr, of wheat atg6s responding to bgt infection.
in the broad-spectrum resistance gene pm21- and the isolate-specific resistance gene pm3f-triggered resistance responses, the expression of wheat atg6s was upregulated by bgt, producing bimodal profiles over a time interval of  <dig> to 36 h after inoculation  with bgt .
the two peaks of transcript accumulation occurred at  <dig> and  <dig> hai.
upon bgt challenge, pm <dig> activated the three atg6s to about the same extent, while pm3f activated taatg6a to a greater extent than taatg6b and 6c.
in the susceptible response of yangmai  <dig> to bgt, however, a single instance of upregulated expression within  <dig> to  <dig> hai was detected for the three atg6s .
this corresponded to, but was higher than, the second instance of upregulated expression detected in the two resistance responses.
in the susceptible response of chancellor to bgt, only one member, taatg6a, showed a bgt-induced expression profile .
these bgt-upregulated expression patterns implied that taatg6s are involved in the wheat immune response to powdery mildew.
wheat atg6s play a weak, positive role in the pm21-triggered resistance response to bgt
we used taatg <dig> knockdown plants to test if taatg6s have any effect on bgt infection.
to exclude the influence of early senescence/chlorosis on bgt infection, only those knockdown plants that did not show obvious early senescence and chlorosis were selected for resistance evaluation.
bsmv-treated plants were heavily inoculated with bgt conidia, and disease symptoms were determined when mildew colonies were apparently visible on susceptible plant leaves.
the knocking down of taatg6s did not result in visible mildew colonies on leaves of bsmv:atg6-treated 92r137/yangmai  <dig>  plants by  <dig> dai, which seemed to retain a resistant phenotype similar to bsmv:gfp-treated control plants.
we then stained leaf samples with trypan blue and, under a microscope, counted the number of successfully penetrating bgt conidium with secondary hyphae.
as shown in figure 6a and b, the percentage of successfully penetrating bgt conidia  was very low  on leaves of the highly resistant bsmv:gfp-treated control plants, but significantly increased to  <dig> – <dig> % on leaves of different bsmv:atg6-treated plants.
furthermore, successfully penetrating bgt conidia on leaves of bsmv:atg6-treated plants developed an average of  <dig>  ±  <dig>  prolonged secondary hyphae, whereas only one short secondary hypha was associated with successfully penetrating bgt conidia on leaves of bsmv:gfp-treated control plants .
these results indicated that knocking down taatg6s resulted in a compromised bgt resistance on 92r137/yangmai  <dig>  plants.
therefore, we propose that taatg6s play a positive role in pm21-triggered wheat resistance response to bgt and that this positive role is weak since no large, sporulating mycelia were observed on taatg <dig> knockdown plants of 92r137/yangmai  <dig>  by  <dig> dai.
in atg <dig>  vps <dig>  atg <dig>  or atg <dig> knockdown tobacco plants and in atg <dig> knockdown or atg <dig> mutant arabidopsis plants, avirulent viral or bacterial pathogen-induced hr-pcd was unrestricted and spread into uninfected cells, suggesting that autophagy has a pro-survival function in the pathogen-induced cell death response  <cit> .
in our study, however, such an unrestricted spread of cell death was not induced by the biotrophic fungal pathogen bgt on taatg <dig> knockdown plants of 92r137/yangmai  <dig> .figure  <dig>
knocking down wheat autophagy-related atg <dig> genes weakly compromises the broad-spectrum resistance gene
pm21
-triggered resistance response to
blumeria graminis
f. sp.
tritici
.
barley stripe mosaic virus -treated 92r137/yangmai  <dig>  plants of the six-leaf stage were heavily inoculated with bgt conidia.
fungal structures were stained by trypan blue and visualized by microscopy.
bgt penetration frequency.
the number of successfully penetrating conidium with secondary hyphae on the fourth leaves was counted at 7 days after inoculation  with bgt conidia.
values represent the mean percentage of successfully penetrating bgt conidia ± sem calculated from at least  <dig> interaction sites.
ck is from bsmv:gfp-treated 92r137/yangmai  <dig>  control plants.
arabic numbers indicate individual bsmv:atg6-treated 92r137/yangmai  <dig>  plants.
representative images of bgt infection on the fourth leaves of bsmv-treated 92r137/yangmai  <dig>  plants at  <dig> dai.
successfully penetrating conidia with secondary hyphae are indicated by arrows.
wheat atg6s also play a negative role in the basal resistance of plants susceptible to bgt
although the bsmv:atg6-treated plants and bsmv:gfp-treated control plants of yangmai  <dig> were both colonized by bgt at  <dig> dai, the sizes of visible mycelia and their sporulation  were significantly reduced on both lower leaves  and upper leaves  of bsmv:atg6-treated plants.
bgt mycelia were also examined under a microscope after trypan blue staining, and a larger portion of small mycelia and a significantly reduced percentage of mycelia with mature chains of conidia were observed on leaves of bsmv:atg6-treated plants at  <dig> dai .
clearly, a significantly enhanced resistance should be responsible for the retarded mycelial spread and sporulation on taatg <dig> knockdown plants.
we thus proposed that taatg6s play negative roles in the basal resistance of the susceptible line yangmai  <dig> to powdery mildew.
moreover, growth-retarded small mycelia, after a prolonged infection time of 9– <dig> dai, induced lesions and chlorosis around lesions on the middle-to-tip leaf regions of bsmv:atg6-treated yangmai  <dig> plants .
the exhibition of enhanced resistance preceded the occurrence of leaf lesions and not all small mycelia were finally associated with leaf cell death , although leaf cell death may further impair the biotrophic lifestyle of bgt.figure  <dig>
knocking down wheat autophagy-related atg <dig> genes enhances the basal resistance of susceptible plants to
blumeria graminis
f. sp.
tritici
.
barley stripe mosaic virus -treated yangmai  <dig> plants of the six-leaf stage were heavily inoculated with bgt conidia.
representative images of mildew colonies on the fourth leaves of bsmv-treated yangmai  <dig> plants.
images were taken at 5 days after inoculation  with bgt conidia.
sporulation of bgt colonies on bsmv-treated yangmai  <dig> plants.
leaf discs with visible mildew colonies were gently harvested from different regions of the fourth leaves at  <dig> dai, immersed in  <dig> % glycerol and subjected to ultrasonic treatment for 1 min to disperse the conidia.
the conidial production per mildew colony was counted by microscopic observation.
values represent the mean conidial production per mildew colony ± sd of at least  <dig> mildew colonies from five plants.
asterisks indicate a significant difference  between the bsmv:atg6-treated plants and bsmv:gfp-treated control plants of yangmai  <dig>   percentage of mycelia with mature chains of conidia on bsmv-treated yangmai  <dig> plants.
fungal structures were stained by trypan blue and visualized by light microscopy.
the number of mycelium with mature chains of conidia on the fourth leaves was counted at  <dig> dai.
at least  <dig> mycelia from five individual plants were counted and values represent the mean percentage of mycelia with mature conidia ± sem.
lesions and chlorosis are induced by
blumeria graminis
f. sp.
tritici
on wheat autophagy-related
atg6
knockdown susceptible plants..  representative images of mildew colonies, leaf lesions and chlorosis on the sixth leaves of bsmv-treated yangmai  <dig> plants at 10 days after inoculation  with bgt conidia.
microscopic observation of growth-retarded small mycelia associated  or not associated  with leaf cell death on bsmv:atg6-treated yangmai  <dig> plants at  <dig> dai.
discussion
as a key component of the class iii pi3k kinase complex, atg <dig> is essential for autophagy and the pi3p-signaling pathways.
previously, plant atg <dig> orthologs had been identified in nicotiana benthamiana, arabidopsis and rice, and their involvement in autophagy had been demonstrated  <cit> .
here, we characterized three wheat atg <dig> genes, taatg6a, 6b and 6c, which were localized on the homeologous chromosomes 3dl, 3bl and 3al, respectively, of the allo-hexaploid wheat genome .
heterologous expression of taatg6a, 6b, or 6c can restore autophagy in atg6-mutant yeast cells.
the taatg6a-gfp fusion protein was located in cytoplasmic punctate pas structures.
constitutive and pathogen-induced autophagy processes were impaired in taatg <dig> knockdown plants.
collectively, these results support the essential role of taatg6s in wheat autophagy biogenesis.
consistent with arabidopsis plants deficient in the pi3k components atg <dig> or vps <dig>  <cit> , taatg <dig> knockdown wheat plants exhibit abnormal growth phenotypes, such as dwarfing, chlorosis, and accelerated senescence/death, under normal conditions.
however, the role of pi3k on normal plant growth may be beyond autophagy, as mutants of autophagy-specific atg generally do not show such extensive growth abnormalities under normal conditions  <cit> .
pathogen-induced expression patterns and positive roles of atg <dig> in plant innate immunity have been reported in plant pathosystems, such as n. benthamiana-tmv  <cit>  and arabidopsis-pseudomonas syringae  <cit> .
here, two transcript accumulation events, an early and a late one, of wheat atg6s were induced in the pm21- and pm3f-triggered wheat resistance responses to bgt within  <dig> to  <dig> hai.
bgt-induced bimodal expression profiles have also been characterized for other powdery mildew resistance-related genes in wheat  <cit> .
two pathogen-induced oxygen burst processes reported previously also match the two time points  <cit> .
the early wheat atg <dig> transcript accumulation event occurred within  <dig> to  <dig> hai, which is when bgt appressorium germ tubes contact and attempt to invade wheat epidermal cells.
this transcript accumulation may be pivotal for the pm21-triggered resistance response because it was not detected in the isogenic susceptible plants of yangmai  <dig>  although knocking down taatg6s did not result in a striking change in the pm21-triggered resistance phenotype, an increased penetration frequency of inoculated bgt conidia was observed by microscopy on knockdown plants of 92r137/yangmai  <dig> .
since successfully penetrating conidia on knockdown plants were inevitably arrested before extending into large mycelia, we hypothesize that taatg6s play only a weak positive role, probably in the early phase of bgt invasion, in the pm21-triggered resistance response.
recent studies on autophagy-specific atg gene mutants support the involvement of autophagy in plant immune responses  <cit> .
the bgt-enhanced occurrence of lysotracker red-stained autolysosome-like structures in control plants of 92r137/yangmai  <dig> , which “disappeared” in taatg <dig> knockdown plants, also links autophagy to atg <dig> during the wheat immune response to bgt.
positive roles of autophagy in plant resistance responses have been reported  <cit> .
similar to our finding of a weak, positive role of taatg6s in the pm21-triggered resistance response to bgt, arabidopsis atg <dig> and atg <dig> mutants are compromised in the early restriction of avirulent p. syringae growth, but retain the capacity to induce other defense responses  <cit> .
contrary to their positive roles in the pm21-triggered resistance response, taatg6s had negative roles in the basal resistance of the susceptible line yangmai  <dig>  knocking down taatg6s enhanced the resistance of yangmai  <dig> plants to bgt mycelial extension and sporulation.
our expression data showed that, compared with their performances in resistance plants of 92r137/yangmai  <dig> , taatg6s were absent for the first response to bgt, which occurred within 0– <dig> hai, but were intensely involved in the second response to bgt, which occurred after  <dig> hai, in the susceptible plants of yangmai  <dig>  the large quantity of successfully penetrating bgt conidia on susceptible plants may induce the highly upregulated expression of taatg6s after  <dig> hai.
the retarded mycelial spread and sporulation on taatg <dig> knockdown susceptible plants suggested that, in wild-type susceptible plants, the atg <dig> recruitment after  <dig> hai might be beneficial for the further development of bgt mycelium.
thus, high levels of taatg6s, and certain taatg6-regulated pathways, in susceptible host cells might be hijacked by bgt after the penetration stage to facilitate its pathogenicity, leading them to function negatively in the basal resistance of susceptible plants.
the autophagy process in host cells is a potential target because of its function in the remobilization of cellular nutrients, which may be exploited by bgt to meet the nutrient supply for fungus proliferation.
negative roles of autophagy in plant basal resistance have been reported in the susceptible responses of arabidopsis to the powdery mildew fungus golovinomyces cichoracearum  <cit>  and to the bacterial pathogen p. syringae  <cit> .
however, we failed to observe autophagy activity in bgt-infected cells of wild-type yangmai  <dig> plants using the lysotracker red staining method, probably because of the very low induction level .
considering this and the pleiotropy of atg <dig>  we cannot exclude other atg6- or pi3p-regulated pathways, or their interplay with autophagy, in underpinning the negative role of atg <dig> in the basal resistance of susceptible wheat plants to powdery mildew.
cell death was induced by growth-retarded small mycelia of bgt on the leaves of taatg <dig> knockdown plants of yangmai  <dig>  however, cell death does not seem to be the reason for the enhanced resistance because it occurred after the exhibition of enhanced resistance and was not induced by all small mycelia.
similarly, powdery mildew induced lesions, but they were not necessarily coupled with the enhanced resistance of susceptible arabidopsis atg <dig> mutants  <cit> .
the enhanced resistance of arabidopsis pmr mutants to the powdery mildew pathogen erysiphe cichoracearum and autophagy-defective mutants to p. syringae is also not associated with cell death  <cit> .
since atg <dig> is autophagy-specific, the powdery mildew-induced cell death of susceptible arabidopsis atg <dig> mutants indicates a pro-survival role of autophagy in this compatible interaction  <cit> .
however, in view of the weak bgt-induced autophagy level and the pleiotropy of atg <dig>  whether autophagy alone or combined with other atg6-regulated pathways underlies the suppression function of taatg6s on bgt-induced cell death of susceptible wheat plants needs to be determined.
CONCLUSIONS
in this study we provide evidence from yeast mutant complementation, subcellular localization of gfp fusions, and detection of autophagy levels in knockdown plants to show that the three identified wheat atg6s are essential for autophagy biogenesis.
bgt-induced expression patterns, and the altered symptoms of powdery mildew on knockdown plants, suggest that taatg6s are implicated in wheat immunity to bgt, playing a weak, positive role in the pm21-triggered resistance response and a negative role in the basal resistance of the susceptible line yangmai <dig>  these findings lay the foundations for a better understanding of autophagy mechanisms in crop plants and the role of atg <dig> in wheat immunity.
additional files
additional file 1: figure s <dig>   sequence comparison and phylogenetic relationship of eukaryotic autophagy-related atg <dig> proteins.
sequence comparison between wheat atg <dig> proteins and their homologs.
the predicted amino acid sequences of wheat taatg6a, 6b and 6c were aligned with homologous atg6s from arabidopsis , yeast  and human .
the alignment was generated in clustalx  <dig>  and viewed in the genedoc  <dig>  program.
numbers on the right indicate amino acid residue positions.
similarity was coded as follows: 100%, black;  <dig> to 100%, dark gray;  <dig> to 80%, light gray; and < 60%, white.
the atg <dig> domain  region is under the straight lines.
phylogenetic relationship of plant atg6s.
the phylogenetic tree was generated with mega  <dig> using the neighbor-joining method.
the reliability of internal branches was assessed by bootstrapping, with  <dig> bootstrap replicates, and the values are shown in percentages, with a branching cut-off at 50%.
wheat atg6s are indicated by diamonds.
figure s <dig>  diagram of the exon-intron structures of wheat atg <dig> genomic orf sequences.
exons  and introns  are drawn to scale, except introns indicated by question marks, which represent there are gaps between available genomic sequences.
two vertical short lines in intron  <dig> of taatg6b represent direct short sequence repeats  delimiting the non-ltr retrotransposon  sequence.
additional file 2: table s <dig>   list of primers used in this paper.
abbreviations
atgautophagy-related gene
bsmvbarley stripe mosaic virus
gfpgreen fluorescent protein
paspre-autophagosomal structure/phagophore assembly site
pi3kphosphatidylinositol 3-kinase
vigsvirus-induced gene silencing
jieyu yue and hong sun contributed equally to this work.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
jy and hw designed the research.
jy, hs, wz, dp and yh conducted the experiments and analyzed the data.
hw wrote the manuscript.
all authors read and approved the final manuscript.

 unlike animals, plants are sessile and must overcome or endure variable, sometimes severe environmental conditions. for example, plants have evolved two main defense mechanisms, pathogen-associated molecular pattern -triggered immunity and effecter-triggered immunity, to combat pathogen infections. eti is a much stronger reaction than pti and is often accompanied by a localized hypersensitive response, a type of programmed cell death, to capture the pathogen at the site of infection. autophagy is an evolutionarily conserved, eukaryotic process by which organelles and cytosolic macromolecules are consumed in lysosomes for nutrient recycling. recent evidence from autophagy-defective arabidopsis plants suggests an important role for autophagy in regulating plant immune responses. this role, however, remains unclear since both positive and negative effects of autophagy on plant immunity have been described in different arabidopsis-pathogen systems as well as in the same arabidopsis-pathogen system. likewise, both pro-survival and pro-death roles of autophagy in the regulation of hr-pcd or pathogen-induced cell death have been reported. therefore, the mechanisms behind the relationship between autophagy and plant immunity need to be elucidated in detail when considering plant species other than arabidopsis and more plant-pathogen systems. during autophagy, double-membrane structures called autophagosomes facilitate the transport of cellular cargos into lysosomes for degradation. to date, more than autophagy-related proteins functioning in autophagy induction and regulation, the initiation and biogenesis of autophagosomes, and the fusion of autophagosomes with lysosomes have been identified in yeast and some orthologs identified in animals and plants. in yeast, a class iii phosphatidylinositol 3-kinase complex resides in the pre-autophagosomal structure/phagophore assembly site where it catalyzes phosphatidylinositol 3-phosphate synthesis and recruits pi3p-binding proteins, especially the atg18-atg complex, for the initiation of autophagic membranes. yeast atg6/vacuolar protein sorting, the ortholog of mammalian beclin is the core component of the pi3k complex along with vps vps and atg and is essential for auophagy. plant homologs of yeast atg play similar essential roles in autophagy, for atg6-deficient plants produce a reduced number of autolysosomes under autophagy-inducing conditions, and the expression of plant atg restores autophagy in atg6-mutant yeast. atg6-deficient plants, like mutants of other atg genes, show an enhanced sensitivity to nutrient starvation and other stress factors. this enhanced sensitivity may result from impaired autophagy because autophagy is responsible for nutrient recycling and distribution under severe environmental conditions. however, the atg6-associated pi3k complex is not autophagy-specific. its phospholipid product, pi3p, is a well-known second messenger involved in receptor signaling and protein sorting-related vesicle trafficking events. homozygous plant mutants of atg vps or vps cannot be obtained because of the requirement of pi3k in pollen development, germination and pollen tube growth. such effects may be attributed to the pi3p-, or its derivative pip2-, signaling pathways rather than to autophagy because homozygous mutants of the autophagy-specific atg genes can be routinely obtained by selfing heterozygous individuals. atg6-deficient plants also show more severe growth abnormalities under normal conditions than mutants of the autophagy-specific atg genes, although they have similarly reduced autophagy levels. atg like autophagy-specific factors, is implicated in plant immune responses. yet the pleiotropy of atg makes it difficult to interpret whether autophagy, the pi3p-signaling pathways, or their interplay underlines the roles of atg in plant immunity. well-established roles of atg other than autophagy, also include its involvement in the vacuolar protein sorting pathway of yeast and in tumor suppression through interactions with the anti-apoptotic bcl-2-like proteins in mammals. so far, few studies on the mechanisms and physiological functions of autophagy and other atg6-associated pathways have been reported in crop plants, and fewer still in common wheat, which is an important food crop worldwide. here, we report the identification of three members of the wheat atg family. the conserved function of wheat atg6s in autophagy was shown using yeast functional complementation, subcellular localization of green fluorescent protein fusions and the determination of autophagy levels in atg knockdown plants. a role for atg in the wheat immune response to the causal fungus, blumeria graminis f. sp. tritici, of powdery mildew was established by bgt-induced atg expression profiles and by altered powdery mildew symptoms on atg knockdown plants. methods plant materials and fungal strain wheat used in this study included isogenic lines 92r137/yangmai and yangmai and isogenic lines michigan amber/chancellor and chancellor. 92r137/yangmai carries the broad-spectrum powdery mildew resistance gene pm michigan amber/chancellor carries the isolate-specific powdery mildew resistance gene pm3f. yangmai and chancellor are susceptible to powdery mildew. the prevalent chinese bgt isolate e which is avirulent to pm and pm3f, was maintained on seedlings of the susceptible wheat cultivar sumai under spore-proof conditions. plant growth conditions and treatments all wheat plants were cultured hydroponically in one-fifth strength hoagland’s solution under normal conditions of 22°c/18°c and a photoperiod of 16 h light/8 h dark in controlled climate chambers. two-leaf stage seedlings were subjected to different treatments. pathogen inoculation was conducted by vigorously shaking off fresh bgt conidia from diseased sumai plants onto experimental seedlings. for abiotic stress treatments, seedlings were transferred to the same one-fifth strength hoagland’s solution containing 200 mm nacl or 16% peg. additional abiotic stress treatments were nitrogen deprivation, and low temperature combined with darkness, in which seedlings were transferred to a 4°c refrigerator and kept in the dark. leaves harvested at defined time points were frozen with liquid nitrogen and then stored at −80°c. these leaf samples were used for gene isolation and expression analyses. isolation of wheat atg genes and expression analyses total rna was prepared from each harvested leaf sample using trizol and subjected to rnase-free dnase i digestion and purification. first-strand cdna was synthesized using oligo primers and the quantscript rt kit. the protein sequence of arabidopsis atg was used as the input to search for wheat expressed sequence tags in the dbest division of genbank by tblastn. both 5ʹ- and 3ʹ-terminal ests were iteratively used as queries to search for new terminal ests to cover full-length open reading frames. est contigs were assembled in vector nti advance, and their sequences were used to design primers flanking full-length orfs. atg genes were amplified from first-strand cdna using the high-fidelity pfu dna polymerase. amplified products were a-tailed and then cloned into the vector pgem®-t easy for sequencing. sequence data were deposited in the genbank database. the expression patterns of wheat atg6s responding to biotic and abiotic treatments were quantified by quantitative real-time pcr with gene-specific primers. qrt-pcr was conducted in a 20 μl realmastermix mixture containing 1 μl first-strand cdna as the template, and the reaction was performed on an iq. each cdna sample was analyzed in triplicate. relative expression levels were calculated using the 2-ΔΔct method with the amplicon of the wheat β-tubulin gene as an internal control. chromosomal localization of wheat atg6s wheat nullisomic-tetrasomic lines were used to map atg genes on specific chromosomes. dna of each line was isolated and used as the template for pcr amplification with gene-specific primers. amplified products were analyzed by 12% polyacrylamide gel electrophoresis. chromosome localization was inferred based on the absence of amplified fragments from lines lacking specific chromosomes. complementation test of yeast atg mutant the orf of each wheat atg cdna was amplified as a noti- noti fragment using the primers listed in additional file 2: table s amplified products were digested and cloned into the yeast expression vector pfl in which the constitutive expression of the inserted gene was driven by the phosphoglycinecerate kinase gene promoter. constructed vectors were sequenced to confirm the correct cdna insertion direction. wild-type yeast strain by and a mutant strain, atg, were purchased from open biosystems. each constructed vector was introduced into yeast atg cells according to the liac/ss-dna/peg trafo protocol. positive transformants were screened on sc-u medium. the yeast autophagy complementation test was performed as previously described. the accumulation of autophagic bodies in vacuoles was observed under a differential interference contrast microscope. subcellular localization of wheat atg6s the orf of each wheat atg cdna lacking a stop codon was amplified as an xhoi-spei fragment using primers listed in additional file 2: table s amplified products were digested, and ligated upstream and in-frame with the orf of gfp in the vector pa7-gfp, in which the gfp fusion construct’s expression was driven by two tandem 35s promoters. prepared plasmids were introduced into onion epidermal cells by particle bombardment with pds1000/he. gfp fluorescence was visualized by epifluorescence microscopy 24 h after bombardment. virus-induced gene silencing a barley stripe mosaic virus -based vigs method was used to create gene knockdown plants. to maximize knockdown efficiency, three bsmvγ vectors, each containing a 200–241 bp fragment from the upstream, middle, or downstream region of the taatg6a coding sequence, were constructed, and in vitro transcribed rnaγ molecules from these vectors were premixed for virus infection. to circumvent the potential functional redundancy among paralogs, we intended to produce simultaneous knockdowns of wheat atg members by choosing fragments from taatg6a for the construction of bsmvγ vectors with – % sequence identities to the corresponding fragments of taatg6b and 6c and multiple nucleotide stretches longer than 23 bp that are 100% identical among the three atg members. the three fragments were prepared by pcr using the primers containing nhei sites listed in additional file 2: table s each amplified fragment was digested by nhei and then inserted in the reverse orientation into the vector bsmvγ to produce the vectors bsmvγ:taatg6as- bsmvγ:taatg6as- and bsmvγ:taatg6as- plasmids of these three bsmvγ derivatives, as well as bsmvγ:gfp and bsmvα, were linearized by mlui, and bsmvβ was linearized by spei. linearized vectors were in vitro transcribed to produce 5ʹ-capped infectious bsmv rna molecules using the ribomax large scale rna production-t kit with the addition of a cap analog in the transcription mixture. rnaγ:taatg6as- rnaγ:taatg6as- and rnaγ:taatg6as- were premixed in a 1:1: ratio to produce rnaγ:taatg6as. the second fully expanded leaves of wheat seedlings at the two-leaf stage were mechanically infected with a 1:1: mixture of rnaα, rnaβ and rnaγ:taatg6as in 1× gkp buffer. seedlings infected with a 1:1: mixture of rnaα, rnaβ and rnaγ:gfp in 1× gkp buffer were used as controls. three replications of the vigs experiment were conducted on the two isogenic lines of 92r137/yangmai and yangmai in each replication, a total of seedlings per line were used for each of the bsmv:atg and bsmv:gfp treatments. the bsmv-treated plants were maintained on one-fifth strength hoagland’s solution under normal conditions. the success of the virus infection was judged by visual observations of mild chlorotic mosaic symptoms on the third and upper leaves of bsmv-treated plants. to confirm the knockdown efficiency, eight plants with virus symptoms were randomly selected from each of the two bsmv treatments, and their transcript levels of taatg6s in the fourth leaves were determined using qrt-pcr as described above with a primer pair designed to conserve regions in the three wheat atg6s. determination of autophagy level by lysotracker red staining bsmv-treated plants at the four-leaf stage were inoculated with bgt conidia as described above. the fourth leaves were excised at and 7 days after inoculation with bgt, infiltrated with 100 μm e-64d and then stained with 2 μm lysotracker red dnd- according to the procedure described previously. fluorescence signals were observed under a confocal microscope using a 543-nm excitation laser and to 615-nm band pass. chlorophyll content determination the fifth leaves of bsmv-treated plants at the five-leaf stage were excised, weighed and ground in 1 ml of 80% acetone. the chlorophyll content was determined spectrophotometrically according to patel and dinesh-kumar. leaf senescence assay green sections were excised from the fourth leaves of bsmv-treated plants at the four-leaf stage and floated on deionized water at room temperature in the dark. leaf sections were photographed every day after dark incubation. the experiment was repeated in triplicate using leaves of three or more plants in each experiment. evaluation of powdery mildew resistance to exclude the influence of early senescence/chlorosis on bgt infections, only those bsmv:atg6-treated plants without obvious symptoms of early senescence and chlorosis were selected for resistance evaluation. bsmv-treated plants at the six-leaf stage were inoculated with bgt conidia as described above. for bsmv-treated yangmai plants, leaf discs with visible mildew colonies were gently harvested from different regions of the fourth leaves at dai, immersed in % glycerol and subjected to an ultrasonic treatment for 1 min to disperse the conidia. the conidial production per mildew colony was counted by microscopic observation and expressed as the mean ± sd of at least mildew colonies from five plants. fungal structures were stained by trypan blue and visualized by light microscopy. for bsmv-treated 92r137/yangmai plants, the number of successfully penetrating conidium with secondary hyphae on the fourth leaves was counted at dai and expressed as a percentage of at least interaction sites on individual plants. the number of secondary hypha associated with each successfully penetrating conidium was also counted and expressed as the mean ± sd of the observed successfully penetrating conidia on all of the investigated plants. for bsmv-treated yangmai plants, the number of mycelium with mature chains of conidia on the fourth leaves was counted at dai and expressed as a percentage of at least mycelia on the leaves of five plants. statistical analysis all experiments were repeated three times with similar results. quantitative data were statistically analyzed using student’s t test using the ibm spss software package.  isolation of atg homologs from wheat using the sequence of arabidopsis atg as a query, putative wheat atg6-encoding ests were mined from the dbest division of genbank and assembled into contigs. primers covering the full-length orfs of these contigs were designed and used for gene isolation by pcr from a leaf cdna pool of the 92r137/yangmai plants induced by bgt for 48 h. three genes, which encoded a typical atg domain, were identified as atg homologs in wheat and designated as taatg6a, 6b and 6c. the orf nucleotide sequences of taatg6a, 6b and 6c share identities of – % and encode peptide sequences of and amino acids, respectively, with 98–99% similarity. in the eukaryotic atg phylogenetic tree, plant atg6s are separately grouped into monocot and dicot clades. taatg6a-6c showed 90–93% sequence similarities to osatg6a-6c from rice and 75% to atatg from arabidopsis. the atg domain region is more conserved across all plant atg6s, whereas the n- and c-terminal flanking sequences show divergence between the monocot and dicot atg6s. two methods, gene-specific pcr amplification of wheat ntl dnas and blast searches against the mapped wheat genomic sequences, were performed to determine the chromosomal localizations of wheat atg members. taatg6b and 6c were localized on chromosomes 3bl and 3al, respectively, by both methods. taatg6a was localized on chromosome 3dl based only on the results of blast searches.figure chromosomal localization of wheat autophagy-related atg genes. dna of each wheat nullisomic-tetrasomic line was used as a template for pcr amplification with gene-specific primers. amplified products were analyzed on 12% polyacrylamide gels. chromosome localization was inferred based on the absence of amplified fragments in lines lacking the specific chromosomes. we also obtained the respective genomic sequences of taatg6a, 6b and 6c by blast searches against wheat genomic sequences. similar nine-intron structures of the taatg6a-6c orf sequences were revealed when comparing genomic sequences with the corresponding cdna sequences. however, taatg6b has a much longer intron than taatg6a and 6c. we compared the three genomic sequences and found a 900-bp insertion only in the intron of taatg6b, which is bordered by 19-bp direct short sequence repeats. as it encodes a truncated protein similar to rna-directed dna polymerases and polyproteins of mobile elements and because no long terminal repeats were found around it, this inserted sequence most likely resulted from a transposition event of a non-ltr retrotransposon. a cpg island of 999 bp, covering the start codon, was detected in the taatg6b genomic sequence, which has a % g + c content and a ratio of of the observed versus expected presence of cpg dinucleotides. this cpg island exists in other monocot atg6s as well, including osatg6b from rice, but not in dicot atg6s, such as atatg from arabidopsis. within the cpg island, there is a short gc-rich stretch just downstream of the start codon, and its length varies among paralogs as well as among homologs. only insertions or deletions that involve multiples of three bases are retained in the gc-rich stretch of monocot atg6s because they do not result in translational frame shifts. amino acids encoded in the gc-rich stretch are predominantly glycines and alanines.figure characterization of wheat autophagy-related atg6 sequences. the genomic sequences of monocot atg6s contain a cpg island covering the start codon. the cpg island was predicted using the web tool cpg island searcher with a cpg island standard of longer than 500 bp in length, a minimum g + c content of 55% and a minimum ratio of the observed versus the expected presence of cpg dinucleotides. gray, horizontal lines represent the 1600-bp start codon-covering genomic sequences of taatg6b from wheat, osatg6b from rice and atatg from arabidopsis. short vertical red lines represent cpg dinucleotides. blue horizontal thick lines represent the ranges of predicted cpg islands. short gc-rich stretch just downstream of the start codon in monocot atg sequences. sequences shown are the 5ʹ-terminal cdna sequences beginning with the start codons of monocot atg6s, including taatg6a, 6b and 6c from wheat, am from hordeum vulgare, nm_ ct and nm_ from rice, xm_ from brachypodium distachyon, xm_ from oryza brachyantha, nm_ from zea mays, xm_ and am from sorghum bicolor, am from saccharum officinarum, and xm_ and xm_ from setaria italic. the gc-rich stretches are not properly aligned but the flanking sequences are aligned. the reading frames from atg are indicated by the upper vertical lines. wheat atg6s function in autophagy human and arabidopsis atg can rescue the autophagy activity of atg6-mutant yeast. to determine if wheat atg6s are functional homologs of yeast atg yeast expression vectors were constructed for each taatg and transformed into atg6-mutant yeast cells. autophagic bodies in vacuoles were monitored as an indicator of autophagy activity. after 5 h of nutrient starvation and treatment with the protease inhibitor phenylmethylsulfonyl fluoride, the wild-type yeast cells accumulated numerous autophagic bodies in vacuoles, which were rarely observed in the atg6-mutant yeast cells. cells of atg6-mutant yeast transformed with taatg6a, 6b or 6c partially restored the accumulation of autophagic bodies.figure autophagy-related atg genes function in wheat autophagy. functional complementation of atg6-mutant yeast cells by wheat atg6s. the autophagic bodies accumulated within vacuoles are indicated by arrows. scale bars represent 5 μm. subcellular localization of gfp-taatg fusion structures in onion epidermal cells. scale bars represent 50 μm. localization of gfp-taatg6a in cytoplasmic and perinuclear membrane-like structures in onion epidermal cells. florescence spots localized in cytoplasmic and perinuclear membrane-like structures are indicated by arrows. scale bars represent 10 μm. relative transcript levels of wheat atg6s in leaves of barley stripe mosaic virus -treated plants. the fourth leaves were sampled from bsmv-treated 92r137/yangmai plants under normal conditions and used in the quantification of taatg transcripts by qrt-pcr with a primer pair designed based on identical regions of taatg6a, 6b, and 6c. amplification of the wheat β-tubulin gene served as an internal control. ck is from bsmv:gfp-treated control plants and arabic numbers indicate individual bsmv:atg6-treated plants. constitutive and blumeria graminis f. sp. tritici -induced autophagy are impaired in bsmv:atg6-treated 92r137/yangmai plants. the fourth leaves were excised from bsmv-treated plants at and 7 days after inoculation with bgt conidia, infiltrated with e-64d and then stained with lysotracker red. lysotracker red-stained punctate autolysosome-like structures are indicated by arrows. the results were reproduced from three independent experiments using three or more plants in each experiment. scale bars represent 5 μm. punctate structures of atg6-gfp fluorescence, considered as pas for autophagosome initiation, have been observed in cells of yeast and arabidopsis. here, gfp fusion constructs of wheat atg6s were introduced into onion epidermal cells by particle bombardment. punctate florescence signals were clearly observed in the cytoplasm of cells expressing taatg6a-gfp and seemed not to occur in the central vacuole. fluorescent spots of taatg6a-gfp were sometimes clustered at cytoplasmic and perinuclear membrane-like structures resembling the trans-golgi network and endoplasmic reticulum. localizations of atg6s to endosomes, the trans-golgi network, endoplasmic reticulum, mitochondria, cytoplasm, and nucleus have been reported in yeast and human. unexpectedly, taatg6b- and taatg6c-gfp florescence showed diffused distributions in the cytoplasm and nuclei, which were almost indistinguishable from those in cells expressing gfp alone. therefore, additional signals may be required to trigger the translocation of taatg6b and 6c to pas for their role in autophagy initiation. to determine if an atg deficiency can lead to autophagy impairment in wheat, bsmv:atg6-treated taatg knockdown plants were generated by the vigs method and their autophagy levels were compared with bsmv:gfp-treated control plants. to obtain the maximum knockdown efficiency, bsmv:atg was designed to contain a mix of three bsmv rnaγ molecules, each containing an inserted antisense fragment corresponding to the upstream, middle, and downstream regions of the taatg6a coding sequence. taking into consideration the potential functional redundancy among paralogs in polyploid wheat, bsmv:atg was also designed to knock down all three wheat atg members by inserting three fragments in the rnaγ molecules that are highly conserved among the three genes. as determined by qrt-pcr using a primer pair designed based on identical regions of the three taatg6s, the total constitutive transcript levels of taatg6a-6c were greatly reduced in the bsmv:atg6-treated plants compared with the transcript levels in bsmv:gfp-treated control plants. this high knockdown efficiency was achieved in all eight bsmv:atg6-treated plants investigated, which confirmed the effectiveness of our vigs design. we then inoculated bsmv-treated plants with bgt conidia and determined their constitutive autophagy activity at dai and bgt-induced autophagy activity at dai using the lysotracker red staining method. lysotracker red is a dye that stains acidic organelles, such as lysosomes and endosomes, as well as autophagosomes/autolysosomes. punctate structures labeled by this dye are generally considered indicative of plant autophagy activity. before lysotracker red staining, leaf samples were pre-infiltrated with the cysteine protease inhibitor e-64d to enhance the accumulation of autophagic bodies inside autolysosomes. for the bsmv:gfp-treated control plants of the resistant line 92r137/yangmai, low levels of a lysotracker red-stained punctate pattern were observed at dai, and enhanced levels were observed at dai in bgt-challenged leaf epidermal cells. these bgt-enhanced levels implied that the taatg6s-regulating autophagy process was induced and implicated in the pm21-triggered immune response to powdery mildew. however, because of the knocking down of taatg6s, the lysotracker red-stained punctate structures were not visible in leaf samples of bsmv:atg6-treated plants regardless of bgt challenge. this result of impaired autophagy in taatg knockdown plants, together with the complementation of yeast autophagy-defective cells with each taatg and the observation of pas localization of taatg6a-gfp, demonstrates the essential role of taatg6s in wheat autophagy. wheat atg knockdown plants exhibited growth abnormalities bsmv:atg6-treated plants, when compared with control plants, showed varying degrees of growth defects, including stunted growth and chlorosis under normal conditions. some were thin and weak, and a higher percentage of bsmv:atg6-treated plants shifted into early senescence and accelerated death before the five-leaf stage than of the controls. to further monitor senescence, green leaf sections from plants at the same developmental stage were excised and kept floating on water in the dark to induce senescence. leaf sections from bsmv:atg6-treated plants began senescing within d post-detachment, as indicated by yellowing. this was significantly earlier than leaf sections from control plants, which began senescing by d post-detachment. thus, taatg6s are essential for normal plant growth and to prevent premature senescence in wheat.figure wheat autophagy-related atg6 knockdown plants display growth abnormalities. all barley stripe mosaic virus -treated plants were cultured hydroponically in one-fifth strength hoagland’s solution and kept under normal conditions of 22°c/18°c and a photoperiod of 16 h light/8 h dark in controlled climate chambers. the growth morphology of bsmv-treated yangmai plants. photographs were taken at the three-leaf stage. the second leaves, which had been mechanically infected with bsmv, were generally withered by this stage and thus were excised before photographing. leaf length of bsmv-treated yangmai plants. the lengths of the third leaves were measured at the three-leaf stage. values represent the mean ± sd of data measured from individual plants. asterisks indicate a significant difference between the bsmv:atg6-treated plants and bsmv:gfp-treated control plants. chlorophyll content of bsmv-treated yangmai plants. the chlorophyll content of the fifth leaves was determined at the five-leaf stage. values represent the mean ± sd of data determined from five individual plants. asterisks indicate a significant difference between the bsmv:atg6-treated plants and bsmv:gfp-treated control plants. percentage of bsmv-treated yangmai plants displaying accelerated death before the five-leaf stage. values represent the mean percentage ± sem of at least plants. detached leaves from bsmv:atg6-treated plants display accelerated dark-induced senescence. green sections of the fourth leaves were excised from bsmv-treated yangmai plants at the four-leaf stage and were floated on deionized water at room temperature in the dark. leaf sections were photographed at the indicated days post-detachment. the results are reproduced from three independent experiments using the leaves of three or more plants in each experiment. expression of wheat atg6s in response to abiotic stress factors and fungal infection yangmai plants were studied using qrt-pcr to determine the effects of abiotic stress factors on the expression of wheat atg6s. the expression levels of taatg6b and 6c were upregulated by high salinity, drought, low temperature/darkness and nitrogen deprivation. the expression of taatg6a was also upregulated by high salinity, drought and nitrogen deprivation, but downregulated by low temperature/darkness. among the four investigated abiotic stress factors, wheat atg6s were most sensitive to the two osmotic stress factors, high salinity and drought, with up to 63-fold and 230-fold increases in the accumulation of transcripts. these upregulated expression modes indicate that taatg6s are closely related to the wheat’s responses to abiotic stress factors.figure expression patterns of wheat autophagy-related atg genes. two-leaf stage seedlings were subjected to different treatments. samples were collected at the indicated time points after the initiation of treatment. total rna was extracted and qrt-pcr was performed with gene-specific primers. the relative expression levels were normalized by the wheat β-tubulin gene and relative to the control value measured at 0 h. data represent the mean of three independent experiments ± sd. expression of wheat atg6s in response to abiotic stress factors. yangmai seedlings were transferred to the one-fifth strength hoagland’s solution containing 200 mm nacl or 16% peg. additional seedling treatments were nitrogen deprivation and exposure to 4°c in the dark. expression of wheat atg6s in response to blumeria graminis f. sp. tritici infection. pathogen inoculations were conducted by vigorously shaking fresh bgt conidia from diseased plants onto seedlings of the isogenic lines 92r137/yangmai and yangmai, and the isogenic lines michigan amber/chancellor and chancellor. two powdery mildew resistant lines, 92r137/yangmai and michigan amber/chancellor, and their respective isogenic susceptible lines were included in the expression analysis, using qrt-pcr, of wheat atg6s responding to bgt infection. in the broad-spectrum resistance gene pm21- and the isolate-specific resistance gene pm3f-triggered resistance responses, the expression of wheat atg6s was upregulated by bgt, producing bimodal profiles over a time interval of to 36 h after inoculation with bgt. the two peaks of transcript accumulation occurred at and hai. upon bgt challenge, pm activated the three atg6s to about the same extent, while pm3f activated taatg6a to a greater extent than taatg6b and 6c. in the susceptible response of yangmai to bgt, however, a single instance of upregulated expression within to hai was detected for the three atg6s. this corresponded to, but was higher than, the second instance of upregulated expression detected in the two resistance responses. in the susceptible response of chancellor to bgt, only one member, taatg6a, showed a bgt-induced expression profile. these bgt-upregulated expression patterns implied that taatg6s are involved in the wheat immune response to powdery mildew. wheat atg6s play a weak, positive role in the pm21-triggered resistance response to bgt we used taatg knockdown plants to test if taatg6s have any effect on bgt infection. to exclude the influence of early senescence/chlorosis on bgt infection, only those knockdown plants that did not show obvious early senescence and chlorosis were selected for resistance evaluation. bsmv-treated plants were heavily inoculated with bgt conidia, and disease symptoms were determined when mildew colonies were apparently visible on susceptible plant leaves. the knocking down of taatg6s did not result in visible mildew colonies on leaves of bsmv:atg6-treated 92r137/yangmai plants by dai, which seemed to retain a resistant phenotype similar to bsmv:gfp-treated control plants. we then stained leaf samples with trypan blue and, under a microscope, counted the number of successfully penetrating bgt conidium with secondary hyphae. as shown in figure 6a and b, the percentage of successfully penetrating bgt conidia was very low on leaves of the highly resistant bsmv:gfp-treated control plants, but significantly increased to – % on leaves of different bsmv:atg6-treated plants. furthermore, successfully penetrating bgt conidia on leaves of bsmv:atg6-treated plants developed an average of  ±  prolonged secondary hyphae, whereas only one short secondary hypha was associated with successfully penetrating bgt conidia on leaves of bsmv:gfp-treated control plants. these results indicated that knocking down taatg6s resulted in a compromised bgt resistance on 92r137/yangmai plants. therefore, we propose that taatg6s play a positive role in pm21-triggered wheat resistance response to bgt and that this positive role is weak since no large, sporulating mycelia were observed on taatg knockdown plants of 92r137/yangmai by dai. in atg vps atg or atg knockdown tobacco plants and in atg knockdown or atg mutant arabidopsis plants, avirulent viral or bacterial pathogen-induced hr-pcd was unrestricted and spread into uninfected cells, suggesting that autophagy has a pro-survival function in the pathogen-induced cell death response. in our study, however, such an unrestricted spread of cell death was not induced by the biotrophic fungal pathogen bgt on taatg knockdown plants of 92r137/yangmai.figure knocking down wheat autophagy-related atg genes weakly compromises the broad-spectrum resistance gene pm21 -triggered resistance response to blumeria graminis f. sp. tritici. barley stripe mosaic virus -treated 92r137/yangmai plants of the six-leaf stage were heavily inoculated with bgt conidia. fungal structures were stained by trypan blue and visualized by microscopy. bgt penetration frequency. the number of successfully penetrating conidium with secondary hyphae on the fourth leaves was counted at 7 days after inoculation with bgt conidia. values represent the mean percentage of successfully penetrating bgt conidia ± sem calculated from at least interaction sites. ck is from bsmv:gfp-treated 92r137/yangmai control plants. arabic numbers indicate individual bsmv:atg6-treated 92r137/yangmai plants. representative images of bgt infection on the fourth leaves of bsmv-treated 92r137/yangmai plants at dai. successfully penetrating conidia with secondary hyphae are indicated by arrows. wheat atg6s also play a negative role in the basal resistance of plants susceptible to bgt although the bsmv:atg6-treated plants and bsmv:gfp-treated control plants of yangmai were both colonized by bgt at dai, the sizes of visible mycelia and their sporulation were significantly reduced on both lower leaves and upper leaves of bsmv:atg6-treated plants. bgt mycelia were also examined under a microscope after trypan blue staining, and a larger portion of small mycelia and a significantly reduced percentage of mycelia with mature chains of conidia were observed on leaves of bsmv:atg6-treated plants at dai. clearly, a significantly enhanced resistance should be responsible for the retarded mycelial spread and sporulation on taatg knockdown plants. we thus proposed that taatg6s play negative roles in the basal resistance of the susceptible line yangmai to powdery mildew. moreover, growth-retarded small mycelia, after a prolonged infection time of 9– dai, induced lesions and chlorosis around lesions on the middle-to-tip leaf regions of bsmv:atg6-treated yangmai plants. the exhibition of enhanced resistance preceded the occurrence of leaf lesions and not all small mycelia were finally associated with leaf cell death, although leaf cell death may further impair the biotrophic lifestyle of bgt.figure knocking down wheat autophagy-related atg genes enhances the basal resistance of susceptible plants to blumeria graminis f. sp. tritici. barley stripe mosaic virus -treated yangmai plants of the six-leaf stage were heavily inoculated with bgt conidia. representative images of mildew colonies on the fourth leaves of bsmv-treated yangmai plants. images were taken at 5 days after inoculation with bgt conidia. sporulation of bgt colonies on bsmv-treated yangmai plants. leaf discs with visible mildew colonies were gently harvested from different regions of the fourth leaves at dai, immersed in % glycerol and subjected to ultrasonic treatment for 1 min to disperse the conidia. the conidial production per mildew colony was counted by microscopic observation. values represent the mean conidial production per mildew colony ± sd of at least mildew colonies from five plants. asterisks indicate a significant difference between the bsmv:atg6-treated plants and bsmv:gfp-treated control plants of yangmai percentage of mycelia with mature chains of conidia on bsmv-treated yangmai plants. fungal structures were stained by trypan blue and visualized by light microscopy. the number of mycelium with mature chains of conidia on the fourth leaves was counted at dai. at least mycelia from five individual plants were counted and values represent the mean percentage of mycelia with mature conidia ± sem. lesions and chlorosis are induced by blumeria graminis f. sp. tritici on wheat autophagy-related atg6 knockdown susceptible plants.. representative images of mildew colonies, leaf lesions and chlorosis on the sixth leaves of bsmv-treated yangmai plants at 10 days after inoculation with bgt conidia. microscopic observation of growth-retarded small mycelia associated or not associated with leaf cell death on bsmv:atg6-treated yangmai plants at dai. discussion as a key component of the class iii pi3k kinase complex, atg is essential for autophagy and the pi3p-signaling pathways. previously, plant atg orthologs had been identified in nicotiana benthamiana, arabidopsis and rice, and their involvement in autophagy had been demonstrated. here, we characterized three wheat atg genes, taatg6a, 6b and 6c, which were localized on the homeologous chromosomes 3dl, 3bl and 3al, respectively, of the allo-hexaploid wheat genome. heterologous expression of taatg6a, 6b, or 6c can restore autophagy in atg6-mutant yeast cells. the taatg6a-gfp fusion protein was located in cytoplasmic punctate pas structures. constitutive and pathogen-induced autophagy processes were impaired in taatg knockdown plants. collectively, these results support the essential role of taatg6s in wheat autophagy biogenesis. consistent with arabidopsis plants deficient in the pi3k components atg or vps, taatg knockdown wheat plants exhibit abnormal growth phenotypes, such as dwarfing, chlorosis, and accelerated senescence/death, under normal conditions. however, the role of pi3k on normal plant growth may be beyond autophagy, as mutants of autophagy-specific atg generally do not show such extensive growth abnormalities under normal conditions. pathogen-induced expression patterns and positive roles of atg in plant innate immunity have been reported in plant pathosystems, such as n. benthamiana-tmv and arabidopsis-pseudomonas syringae. here, two transcript accumulation events, an early and a late one, of wheat atg6s were induced in the pm21- and pm3f-triggered wheat resistance responses to bgt within to hai. bgt-induced bimodal expression profiles have also been characterized for other powdery mildew resistance-related genes in wheat. two pathogen-induced oxygen burst processes reported previously also match the two time points. the early wheat atg transcript accumulation event occurred within to hai, which is when bgt appressorium germ tubes contact and attempt to invade wheat epidermal cells. this transcript accumulation may be pivotal for the pm21-triggered resistance response because it was not detected in the isogenic susceptible plants of yangmai although knocking down taatg6s did not result in a striking change in the pm21-triggered resistance phenotype, an increased penetration frequency of inoculated bgt conidia was observed by microscopy on knockdown plants of 92r137/yangmai. since successfully penetrating conidia on knockdown plants were inevitably arrested before extending into large mycelia, we hypothesize that taatg6s play only a weak positive role, probably in the early phase of bgt invasion, in the pm21-triggered resistance response. recent studies on autophagy-specific atg gene mutants support the involvement of autophagy in plant immune responses. the bgt-enhanced occurrence of lysotracker red-stained autolysosome-like structures in control plants of 92r137/yangmai, which “disappeared” in taatg knockdown plants, also links autophagy to atg during the wheat immune response to bgt. positive roles of autophagy in plant resistance responses have been reported. similar to our finding of a weak, positive role of taatg6s in the pm21-triggered resistance response to bgt, arabidopsis atg and atg mutants are compromised in the early restriction of avirulent p. syringae growth, but retain the capacity to induce other defense responses. contrary to their positive roles in the pm21-triggered resistance response, taatg6s had negative roles in the basal resistance of the susceptible line yangmai knocking down taatg6s enhanced the resistance of yangmai plants to bgt mycelial extension and sporulation. our expression data showed that, compared with their performances in resistance plants of 92r137/yangmai, taatg6s were absent for the first response to bgt, which occurred within 0– hai, but were intensely involved in the second response to bgt, which occurred after hai, in the susceptible plants of yangmai the large quantity of successfully penetrating bgt conidia on susceptible plants may induce the highly upregulated expression of taatg6s after hai. the retarded mycelial spread and sporulation on taatg knockdown susceptible plants suggested that, in wild-type susceptible plants, the atg recruitment after hai might be beneficial for the further development of bgt mycelium. thus, high levels of taatg6s, and certain taatg6-regulated pathways, in susceptible host cells might be hijacked by bgt after the penetration stage to facilitate its pathogenicity, leading them to function negatively in the basal resistance of susceptible plants. the autophagy process in host cells is a potential target because of its function in the remobilization of cellular nutrients, which may be exploited by bgt to meet the nutrient supply for fungus proliferation. negative roles of autophagy in plant basal resistance have been reported in the susceptible responses of arabidopsis to the powdery mildew fungus golovinomyces cichoracearum and to the bacterial pathogen p. syringae. however, we failed to observe autophagy activity in bgt-infected cells of wild-type yangmai plants using the lysotracker red staining method, probably because of the very low induction level. considering this and the pleiotropy of atg we cannot exclude other atg6- or pi3p-regulated pathways, or their interplay with autophagy, in underpinning the negative role of atg in the basal resistance of susceptible wheat plants to powdery mildew. cell death was induced by growth-retarded small mycelia of bgt on the leaves of taatg knockdown plants of yangmai however, cell death does not seem to be the reason for the enhanced resistance because it occurred after the exhibition of enhanced resistance and was not induced by all small mycelia. similarly, powdery mildew induced lesions, but they were not necessarily coupled with the enhanced resistance of susceptible arabidopsis atg mutants. the enhanced resistance of arabidopsis pmr mutants to the powdery mildew pathogen erysiphe cichoracearum and autophagy-defective mutants to p. syringae is also not associated with cell death. since atg is autophagy-specific, the powdery mildew-induced cell death of susceptible arabidopsis atg mutants indicates a pro-survival role of autophagy in this compatible interaction. however, in view of the weak bgt-induced autophagy level and the pleiotropy of atg whether autophagy alone or combined with other atg6-regulated pathways underlies the suppression function of taatg6s on bgt-induced cell death of susceptible wheat plants needs to be determined.  in this study we provide evidence from yeast mutant complementation, subcellular localization of gfp fusions, and detection of autophagy levels in knockdown plants to show that the three identified wheat atg6s are essential for autophagy biogenesis. bgt-induced expression patterns, and the altered symptoms of powdery mildew on knockdown plants, suggest that taatg6s are implicated in wheat immunity to bgt, playing a weak, positive role in the pm21-triggered resistance response and a negative role in the basal resistance of the susceptible line yangmai these findings lay the foundations for a better understanding of autophagy mechanisms in crop plants and the role of atg in wheat immunity. additional files additional file 1: figure s sequence comparison and phylogenetic relationship of eukaryotic autophagy-related atg proteins. sequence comparison between wheat atg proteins and their homologs. the predicted amino acid sequences of wheat taatg6a, 6b and 6c were aligned with homologous atg6s from arabidopsis, yeast and human. the alignment was generated in clustalx and viewed in the genedoc program. numbers on the right indicate amino acid residue positions. similarity was coded as follows: 100%, black; to 100%, dark gray; to 80%, light gray; and < 60%, white. the atg domain region is under the straight lines. phylogenetic relationship of plant atg6s. the phylogenetic tree was generated with mega using the neighbor-joining method. the reliability of internal branches was assessed by bootstrapping, with bootstrap replicates, and the values are shown in percentages, with a branching cut-off at 50%. wheat atg6s are indicated by diamonds. figure s diagram of the exon-intron structures of wheat atg genomic orf sequences. exons and introns are drawn to scale, except introns indicated by question marks, which represent there are gaps between available genomic sequences. two vertical short lines in intron of taatg6b represent direct short sequence repeats delimiting the non-ltr retrotransposon sequence. additional file 2: table s list of primers used in this paper. abbreviations atgautophagy-related gene bsmvbarley stripe mosaic virus gfpgreen fluorescent protein paspre-autophagosomal structure/phagophore assembly site pi3kphosphatidylinositol 3-kinase vigsvirus-induced gene silencing jieyu yue and hong sun contributed equally to this work. competing interests the authors declare that they have no competing interests. authors’ contributions jy and hw designed the research. jy, hs, wz, dp and yh conducted the experiments and analyzed the data. hw wrote the manuscript. all authors read and approved the final manuscript. 
a role for taatg6s in wheat immunity to powdery mildew was further implied when knockdowns of taatg6s weakly compromised the broad-spectrum powdery mildew resistance gene pm21-triggered resistance response and, conversely and significantly, enhanced the basal resistance of susceptible plants. wheat atg6s are implicated in immunity to powdery mildew, playing a weak, positive role in the pm21-triggered resistance response and a negative role in the basal resistance of susceptible plants. we also examined the expression patterns of wheat atg6s for clues to their physiological roles, and found that their expression was induced by the fungus blumeria graminis f. sp. the expression of each taatg gene restored the accumulation of autophagic bodies in atg6-mutant yeast. arabidopsis atg regulates normal plant growth, pollen development and germination, and plant responses to biotic/abiotic stresses. we identified three members, taatg6a-6c, of the atg family from common wheat. autophagyatg6common wheat powdery mildewissue-copyright-statement© the author 2015 taatg6a, 6b and 6c were localized on homeologous chromosomes 3dl, 3bl and 3al, respectively, of the allo-hexaploid wheat genome, and evidence was provided for their essential role in autophagy. three wheat atg6s were identified and shown to be essential for autophagy biogenesis. in addition, leaf cell death was sometimes induced by growth-retarded small bgt mycelia on susceptible taatg knockdown plants after a long period of interaction. the taatg6a-gfp fusion protein was found in punctate pre-autophagosomal structures. tritici, the causal agent of powdery mildew, and by abiotic stress factors. additionally, taatg knockdown plants showed impaired constitutive and pathogen-induced autophagy and growth abnormalities under normal conditions.
 autophagy-related atg proteins are pleiotropic proteins functioning in autophagy and the phosphatidylinositol 3-phosphate-signaling pathways. arabidopsis atg regulates normal plant growth, pollen development and germination, and plant responses to biotic/abiotic stresses. however, the atg functions in wheat, an important food crop, are lacking.  we identified three members, taatg6a-6c, of the atg family from common wheat. taatg6a, 6b and 6c were localized on homeologous chromosomes 3dl, 3bl and 3al, respectively, of the allo-hexaploid wheat genome, and evidence was provided for their essential role in autophagy. the taatg6a-gfp fusion protein was found in punctate pre-autophagosomal structures. the expression of each taatg gene restored the accumulation of autophagic bodies in atg6-mutant yeast. additionally, taatg knockdown plants showed impaired constitutive and pathogen-induced autophagy and growth abnormalities under normal conditions. we also examined the expression patterns of wheat atg6s for clues to their physiological roles, and found that their expression was induced by the fungus blumeria graminis f. sp. tritici, the causal agent of powdery mildew, and by abiotic stress factors. a role for taatg6s in wheat immunity to powdery mildew was further implied when knockdowns of taatg6s weakly compromised the broad-spectrum powdery mildew resistance gene pm21-triggered resistance response and, conversely and significantly, enhanced the basal resistance of susceptible plants. in addition, leaf cell death was sometimes induced by growth-retarded small bgt mycelia on susceptible taatg knockdown plants after a long period of interaction. thus, we provide an important extension of the previous characterization of plant atg genes in wheat, and observed a role for autophagy genes in wheat immune responses to fungal pathogens.  three wheat atg6s were identified and shown to be essential for autophagy biogenesis. wheat atg6s are implicated in immunity to powdery mildew, playing a weak, positive role in the pm21-triggered resistance response and a negative role in the basal resistance of susceptible plants. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords autophagyatg6common wheat powdery mildewissue-copyright-statement© the author 2015 